GIFT   OF 
MICHAEL  REESE 


BIOLOGY 

LIBRARY 

6 


THE  PRINCIPLES 


OF 


BACTERIOLOGY: 

A  PRACTICAL  MANUAL  FOR  STUDENTS 
AND  PHYSICIANS. 


BY 

A.   C.   ABBOTT,   M.  D., 

\\ 

PROFESSOR  OF  HYGIENE,   AND  DIRECTOR  OF  THE  LABORATORY  OF  HYGIENE, 
UNIVERSITY  OF  PENNSYLVANIA. 

FIFTH  EDITION, 

ENLARGED  AND  THOROUGHLY  REVISED. 
With  109  Illustrations,  of  which  26  are  colored. 


LEA   BROTHERS  &  CO., 
PHILADELPHIA  AND  NEW  YORK. 

1899. 


LIBRARY 
G 


Entered  according  to  Act  of  Congress  in  the  yenr  1899,  by 

LEA   BROTHERS  &   CO., 
in  the  Office  of  the  Librarian  of  Congress.    All  rights  reserved. 


WESTCOTT    4   THOMSON,  ' 

W.   J.   OORNAN,   PHILADA. 


55 

* 


PREFACE  TO   THE  FIFTH  EDITION. 


THE  call  for  the  fifth  edition  of  this  book  within  two 
years  of  the  publication  of  the  fourth  is  naturally  a 
source  of  very  great  gratification  to  the  author,  and  he 
takes  this  occasion  to  express  his  obligations  to  those  of 
his  readers  who  have  materially  contributed  to  the  suc- 
cess of  the  work  by  friendly  comments  and  criticism. 

The  plan  of  this  edition  does  not  differ  from  that 
followed  in  its  predecessors.  Reference  has  been  made 
to  such  investigations  of  the  past  two  years  as  are  per- 
tinent to  a  book  of  this  character,  and  essential  to  an  un- 
derstanding on  the  part  of  the  reader  of  the  lines  along 
which  the  subject  has  advanced.  Several  chapters,  par- 
ticularly those  on  technique,  disinfection,  the  specific 
infections,  and  immunity  have  been  considerably  en- 
larged. In  some  cases  opinions  expressed  in  former  edi- 
tions have  been  modified  to  conform  to  more  advanced 
views.  A  number  of  the  old  illustrations  have  been 
replaced  by  new  ones,  several  of  which  are  in  colors. 
The  author  trusts  that  such  alterations  as  have  been 
made  will  be  regarded  as  adding  to  the  value  of  the 
work. 

PHILADELPHIA,  March,  1899. 


91550 


PREFACE  TO  THE  FIRST  EDITION. 


IN  preparing  this  book  the  author  has  kept  in  mind 
the  needs  of  the  student  and  practitioner  of  medicine, 
for  whom  the  importance  of  an  acquaintance  with  prac- 
tical bacteriology  cannot  be  overestimated. 

It  is  to  advances  made  through  bacteriological  re- 
search that  we  are  indebted  for  much  of  our  knowledge 
of  the  conditions  underlying  infection,  and  for  the  elu- 
cidation of  many  hitherto  obscure  problems  concerning 
the  etiology,  the  modes  of  transmission,  and  the  means 
of  prevention  of  infectious  maladies. 

Only  within  a  comparatively  short  time  have  students 
and  physicians  been  enabled  to  obtain  the  systematic 
instruction  in  this  science  that  is  of  value  in  aiding 
them  in  their  efforts  to  check  disease.  The  rapid  in- 
crease in  the  number  who  are  availing  themselves  of 
these  opportunities  speaks  directly  for  the  practical 
value  of  the  science. 

As  the  majority  of  those  undertaking  the  study  of 
bacteriology  do  so  with  the  view  of  utilizing  it  in  med- 
ical practice,  and  as  many  of  these  can  devote  to  it  but 
a  portion  of  their  time,  it  is  desirable  that  the  subject- 
matter  be  presented  in  as  direct  a  manner  as  possible. 


vi  PREFACE  TO   THE  FIRST  EDITION. 

Presuming  the  reader  to  be  unfamiliar  with  the  sub- 
ject, the  author  has  restricted  himself  to  those  funda- 
mental features  that  are  essential  to  its  understanding. 
The  object  has  been  to  present  the  important  ideas  and 
methods  as  concisely  as  is  compatible  with  clearness, 
and  at  the  same  time  to  accentuate  throughout  the 
underlying  principles  which  govern  the  work. 

With  the  view  of  inducing  independent  thought  on 
the  part  of  the  student,  and  of  diminishing  the  fre- 
quency of  that  oft-heard  query,  "  What  shall  I  do 
next?"  experiments  have  been  suggested  wherever  it 
is  possible.  These  have  been  arranged  to  illustrate  the 
salient  points  of  the  work  and  to  attract  attention  to 
the  minute  details,  upon  the  observation  of  which  so 
much  in  bacteriology  depends. 

PHILADELPHIA,  December,  1891. 


CONTENTS. 


INTRODUCTION. 

PAGES 

"Omne  vivum  ex  vivo" — The  overthrow  of  the  doctrine  of 
spontaneous  generation — Earlier  bacteriological  studies — 
The  birth  of  modern  bacteriology 17-30 

CHAPTER  I.     > 

Definition  of  bacteria — Their  place  in  nature — Difference  be- 
tween parasites  and  saprophytes — Nutrition  of  bacteria — 
Products  of  bacteria — Their  relation  to  oxygen — Influence 
of  temperature  upon  their  growth 31-43 

CHAPTER  II. 

Morphology  of  bacteria — Grouping — Mode  of  multiplication — 
Spore-formation — Motility 44-54 

CHAPTER  III. 

Principles  of  sterilization  by  heat — Methods  employed — Dis- 
continued sterilization — Sterilization  under  pressure — Ap- 
paratus employed— Chemical  disinfection  and  sterilization  .  55-82 

CHAPTER  IV.     ^ 

Principles  involved  in  the  methods  of  isolation  of  bacteria  in 
pure  culture  by  the  plate  method  of  Koch— Materials  em- 
ployed    83-89 

CHAPTER  V. 

Preparation  of  nutrient  media — Bouillon,  gelatin,  agar-agar, 

potato,  blood-serum,  ete 90-119 


Viii  CONTENTS. 

CHAPTER  VI. 

PAGES 

Preparation  of  the  tubes,  flasks,  etc.,  in  which  the  media  are 
to  be  preserved 120-123 

CHAPTER   VII. 

Technique  of  making  plates — Esmarch  tubes,  Petri  plates,  etc.   124-135 

CHAPTER   VIII. 

The  incuba ting-oven — Gas-pressure  regulator — Thermo-regu- 

lator — Safety  burner  employed  in  heating  the  incubator  .    .    136-143 

CHAPTER   IX. 

The  study  of  colonies — Their  naked-eye  peculiarities  and 
their  appearance  under  different  conditions — Differences 
in  the  structure  of  colonies  from  different  species  of  bac- 
teria— Stab-cultures — Slant-cultures  .  144-149 

CHAPTER  X. 

Methods  of  staining — Solutions  employed — Preparation  and 
staining  of  cover-slips — Preparation  of  tissues  for  section- 
cutting — Staining  of  tissues — Special  staining-methods  .  .  150-188 

CHAPTER  XI. 

Systematic  study  of  an  organism — Points  to  be  considered  in 
identifying  an  organism  as  a  definite  species 189-218 

CHAPTER  XII.  •* 

Inoculation  of  animals  —  Subcutaneous  inoculation  —  Intra- 
venous injection — Inoculation  into  the  great  serous  cavities 
and  into  the  anterior  chamber  of  the  eye — Observation  of 
animals  after  inoculation 219-240 

CHAPTER  XIII. 

Post-mortem  examination  of  animals — Bacteriological  exami- 
nation of  the  tissues — Disposal  of  tissues  and  disinfection 
of  instruments  after  the  examination — Study  of  tissues  and 
exudates  during  life 241-250 


CONTENTS.  ix 

APPLICATION  OF   THE  METHODS  OF 
BACTERIOLOGY.      DESCRIPTIONS 
OF  SOME  OF   THE   MORE   IM- 
PORTANT SPECIES. 

CHAPTER  XIV. 

PAGES 

To  obtain  material  with  which  to  begin  work  . 251-254 

CHAPTER  XV. 

Various  experiments  in  sterilization  by  steam  and  by  hot  air  .   255-259 

CHAPTER  XVI. 

Suppuration  —  Staphylococcus  pyoyenes  aureus  —  Staphylococcus 
pyogenes  albus  and  citreiis  —  Streptococcus  pyogenes —  Gono- 
coccus — Bacillus  pyocyaneus — Bacillus  of  bubonic  plague  .  .  260-299 

CHAPTER  XVII. 

Sputum  septicaemia — Septicaemia  resulting  from  the  presence 

of  micrococcus  tetragenus  in  the  tissues 300-311 

CHAPTER  XVIII. 

Tuberculosis— Microscopic  appearance  of  miliary  tubercles — 
Encapsulation  of  tuberculous  foci  —  Diffuse  caseation — 
Cavity-formation — Primary  infection — Modes  of  infection — 
Location  of  the  bacilli  in  the  tissues — Staining-peculiarities 
— Organisms  with  which  bacillus  tuberculosis  may  be  con- 
founded—Points of  differentiation— Bacillus  of  influenza  .  312-338 

CHAPTER  XIX. 

Glanders— Characteristics  of  the  disease— Histological  struct- 
ure of  the  glanders  nodule  —  Susceptibility  of  different 
animals  to  glanders — The  bacillus  of  glanders;  its  morpho- 
logical and  cultural  peculiarities — Diagnosis  of  glanders  .  .  339-348 

CHAPTER  XX. 

Bacillus  diphtheriie — Its  isolation  and  cultivation — Morpho- 
logical and  cultural  peculiarities— Pathogenic  properties- 
Variations  in  virulence— Pseudo-diphtheritic  bacilli  ...  349-368 


x  CONTENTS. 

CHAPTEK  XXI. 

PAGES 

Typhoid  fever — Study  of  the  organism  concerned  in  its  pro- 
duction— Bacterium  coli  commune — Its  resemblance  to  the 
bacillus  of  typhoid  fever — Its  morphological,  cultural,  and 
pathogenic  properties — Its  differentiation  from  bacillus  ty- 
phosus — Methods  of  isolating  the  typhoid  bacillus 369-400 

CHAPTEK  XXII. 

The  spirillum  (comma  bacillus)  of  Asiatic  cholera  —  Its 
morphological  and  cultural  peculiarities — Pathogenic  prop- 
erties— The  bacteriological  diagnosis  of  Asiatic  cholera  .  .  401-428 

CHAPTER  XXIII. 

Organisms  of  interest,  historically  and  otherwise,  that  have 
been  confounded  with  the  spirillum  of  Asiatic  cholera — 
Their  peculiarities  and  differential  features — Vibrio  proteus, 
or  bacillus  of  Finkler  and  Prior—  Spirillum  tyrogenum,  or 
cheese  spirillum  of  Deneke — The  spirillum  of  Miller — Vibrio 
Metchnikovi 429-447 

CHAPTER  XXIV. 

Study  of  bacillus  anthracis,  and  the  effects  produced  by  its  in- 
oculation into  animals — Peculiarities  of  the  organism  under 
varying  conditions  of  surroundings 448-462 

CHAPTER  XXV. 

The  most  important  of  the  organisms  found  in  the  soil — The 
nitrifying  bacteria— The  bacillus  of  tetanus — The  bacillus 
of  malignant  oedema — The  bacillus  of  symptomatic  anthrax  463-488 

CHAPTER  XXVI. 

^J 

Infection  and  immunity — The  types  of  infection ;  intimate 
nature  of  infection— Septicaemia,  toxaemia,  variations  in  in- 
fectious processes — Immunity,  natural  and  acquired — The 
hypotheses  that  have  been  advanced  in  explanation  of  im- 
munity—Conclusions    489-525 

CHAPTER  XXVII. 

Bacteriological  study  of  water  —  Methods  employed  —  Pre- 
cautions to  be  observed — Apparatus  used,  and  methods  of 
using  them — Methods  of  investigating  air  and  soil  ....  526-557 


CONTENTS.  xi 

CHAPTER  XXVIII. 

PAGES 

Methods  of  testing  disinfectants  and  antiseptics — Experiments 
illustrating  the  precautions  to  be  taken — Experiments  in 
skin-disinfection .  558-571 


APPENDIX. 

Apparatus  necessary  in  a  beginner's  bacteriological  laboratory  573-578 


BACTERIOLOGY. 


INTRODUCTION. 

"  Omne  vi vum  ex  vivo  " — The  overthrow  of  the  doctrine  of  spontaneous 
generation— Earlier  bacteriological  studies— The  birth  of  modern 
bacteriology. 

BACTERIOLOGY  may  be  said  to  have  had  its  begin- 
ning with  the  observations  of  Leeuwenhoek  in  the  latter 
part  of  the  seventeenth  century.  Though  its  most  rapid 
and  important  development  has  taken  place  during  the 
past  fifteen  or  twenty  years,  still,  a  review  of  the  vari- 
ous evolutionary  phases  through  which  it  has  passed 
in  the  course  of  more  than  two  hundred  years  re- 
veals an  entertaining  and  instructive  history.  From 
the  very  outset  its  history  is  inseparably  connected 
with  that  of  medicine,  and  to-day  its  relations  to  hy- 
giene and  preventive  medicine  are  of  fundamental  im- 
portance. It  is,  indeed,  through  a  more  intimate  ac- 
quaintance with  the  biological  activities  of  the  unicel- 
lular vegetable  micro-organisms  that  modern  hygiene 
has  attained  the  prominence  and  importance  now  justly 
accorded  to  it.  Through  studies  in  the  domain  of  bac- 
teriology our  knowledge  of  the  causation,  course,  and 
prevention  of  infectious  diseases  is  daily  becoming  more 
accurate,  and  it  is  needless  to  emphasize  the  relation  of 
such  knowledge  to  the  manifold  problems  that  pre- 
sent themselves  to  the  student  of  modern  medicine. 

2  17 


18  BACTERIOLOGY. 

Though  the  contributions  which  have  done  most  to  place 
bacteriology  on  the  footing  of  a  science  are  those  of 
recent  years,  still,  during  the  earlier  stages  of  its  de- 
velopment, many  observations  were  made  which  formed 
the  foundation-work  for  much  that  was  to  follow.  Be- 
fore regularly  beginning  our  studies,  therefore,  it  may 
be  of  advantage  to  acquaint  ourselves  with  the  more 
prominent  of  these  investigations. 

Antony  van  Leeuwenhoek,  the  first  to  describe  the 
bodies  now  recognized  as  bacteria,  was  born  at  Delft,  in 
Holland,  in  1632.  He  was  not  considered  a  man  of 
liberal  education,  having  been  during  his  early  years  an 
apprentice  to  a  linendraper.  During  his  apprenticeship 
he  learned  the  art  of  lens-grinding,  in  which  he  became 
so  proficient  that  he  eventually  perfected  a  simple  lens 
by  means  of  which  he  was  enabled  to  see  objects  of 
much  smaller  dimensions  than  any  hitherto  seen  with 
the  best  compound  microscopes  in  existence  at  that 
date.  At  the  time  of  his  discoveries  he  was  following 
the  trade  of  linendraper  in  Amsterdam. 

In  1675  he  published  the  fact  that  he  had  succeeded 
in  perfecting  a  lens  by  means  of  which  he  could  detect 
in  a  drop  of  rain-water  living,  motile  "  animalcules " 
of  the  most  minute  dimensions — smaller  than  anything 
that  had  hitherto  been  seen.  Encouraged  by  this  dis- 
covery, he  continued  to  examine  various  substances  for 
the  presence  of  what  he  considered  animal  life  in  its 
most  minute  form.  He  found  in  sea-water,  in  well- 
water,  in  the  intestinal  canal  of  frogs  and  birds,  and  in 
his  own  diarrhoeal  evacuations,  objects  that  differenti- 
ated themselves  the  one  from  the  other,  not  only  by 
their  shape  and  size,  but  also  by  the  peculiarity  of 
motility  which  some  of  them  were  seen  to  possess. 


INTRODUCTION.  19 

In  the  year  1683  he  discovered  in  the  tartar  scraped 
from  between  the  teeth  a  form  of  micro-organism  upon 
which  he  laid  special  stress.  This  observation  he  em- 
bodied in  the  form  of  a  contribution  which  was  presented 
to  the  Royal  Society  of  London  on  September  14, 1683. 
This  paper  is  of  particular  importance,  not  only  because 
of  the  careful,  objective  nature  of  the  description  given 
of  the  bodies  seen  by  him,  but  also  for  the  illustrations 
which  accompany  it.  From  a  perusal  of  the  text  and  an 
inspection  of  the  plates  accompanying  it  there  remains 
little  room  for  doubt  that  Leeuwenhoek  saw  with  his 
primitive  lens  the  bodies  now  recognized  as  bacteria.1 

Upon  seeing  these  bodies  he  was  apparently  very 
much  impressed,  for  he  writes :  "  With  the  greatest 
astonishment  I  observed  that  everywhere  throughout 
the  material  which  I  was  examining  were  distributed 
animalcules  of  the  most  microscopic  dimensions,  which 
moved  themselves  about  in  a  remarkably  energetic  way." 

This  discovery  was  shortly  followed  by  others  of  an 
equally  important  nature.  His  field  of  observation 
appears  to  have  increased  rapidly,  for  after  a  time  he 
speaks  of  bodies  of  much  smaller  dimensions  than  those 
at  first  described  by  him. 

Throughout  all  of  Leeuwenhoek's  work  there  is  a 
conspicuous  absence  of  the  speculative.  His  contri- 
butions are  remarkable  for  their  purely  objective 
nature. 

After  the  presence  of  these  organisms  in  water,  in 
the  mouth,  and  in  the  intestinal  evacuations  was  made 
known  to  the  world,  it  is  hardly  surprising  that  they 
were  immediately  seized  upon  as  the  explanation  of  the 

1  See  Arcana  Naturae  detecta  ab  ANTONIO  VAN  LEEUWENHOEK  ; 
Delphis  Batavorum,  1695. 


20  BACTERIOLOGY. 

origin  of  many  obscure  diseases.  So  universal  became 
the  belief  in  a  causal  relation  between  these  "  animal- 
cules "  and  disease  that  it  amounted  almost  to  a  germ- 
mania.  It  became  the  fashion  to  suspect  the  presence 
of  these  organisms  in  all  forms  and  kinds  of  disease, 
simply  because  they  had  been  demonstrated  in  the 
mouth,  intestinal  evacuations,  and  water. 

Though  nothing  of  value  at  the  time  had  been  done 
in  the  way  of  classification,  and  still  less  in  separating 
and  identifying  the  members  of  this  large  group,  still 
the  foremost  men  of  the  day  did  not  hesitate  to  ascribe 
to  them  not  only  the  property  of  producing  pathological 
conditions,  but  some  even  went  so  far  as  to  hold  that 
variations  in  the  appearance  of  symptoms  of  disease 
were  the  result  of  differences  in  the  behavior  of  the 
organisms  in  the  tissues. 

Marcus  Antonius  Plenciz,  a  physician  of  Vienna  in 
1762,  declared  himself  a  firm  believer  in  the  work  of 
Leeuwenhoek,  and  based  the  doctrine  which  he  taught 
upon  the  discoveries  of  the  Dutch  observer  and  upon 
observations  of  a  confirmatory  nature  which  he  himself 
had  made.  The  doctrine  of  Plenciz  assumed  a  causal 
relation  between  the  micro-organisms  discovered  and 
described  by  Leeuwenhoek  and  all  infectious  diseases. 
He  claimed  that  the  material  of  infection  could  be  noth- 
ing else  than  a  living  substance,  and  endeavored  on 
these  grounds  to  explain  the  variations  in  the  period  of 
incubation  of  the  different  infectious  diseases.  He  like- 
wise believed  the  living  contagium  to  be  capable  of 
multiplication  within  the  body,  and  spoke  of  the  possi- 
bility of  its  transmission  through  the  air.  He  claimed 
a  special  germ  for  each  disease,  holding  that  just  as  from 
a  given  cereal  only  one  kind  of  grain  can  grow,  so  by 


INTRODUCTION.  21 

the  special  germ  for  each  disease  only  that  disease  can 
be  produced. 

He  found  in  all  decomposing  matters  innumerable 
minute  "  animalcule,"  and  was  so  firmly  convinced  of 
their  etiological  relation  to  the  process  that  he  formu- 
lated the  law :  that  decomposition  can  only  take  place 
when  the  decomposable  material  becomes  coated  with  a 
layer  of  the  organisms,  and  can  proceed  only  when  they 
increase  and  multiply. 

However  cpnvincing  the  arguments  of  Plenciz  may 
appear,  they  seem  to  have  been  lost  sight  of  in  the 
course  of  subsequent  events,  and  by  a  few  were  even 
regarded  as  the  productions  of  an  unbalanced  mind. 
For  example,  as  late  as  1820  we  find  Ozanam  express- 
ing himself  on  the  subject  as  follows  :  "  Many  authors 
have  written  concerning  the  animal  nature  of  the  conta- 
gion of  infectious  diseases ;  many  have  indeed  assumed 
it  to  be  developed  from  animal  substances  and  that  it 
is  itself  animal  and  possesses  the  property  of  life ;  I 
shall  not  waste  time  in  effort  to  refute  these  absurd 
hypotheses." 

Similar  expressions  of  opinion  were  heard  from  many 
other  medical  men  of  the  time,  all  tending  in  the  same 
direction,  all  doubting  the  possibility  of  these  micro- 
scopic creatures  belonging  to  the  world  of  living  things. 

It  was  not  until  between  the  fourth  and  fifth  decades 
of  the  present  century  that  by  the  fortunate  coincidence 
of  a  number  of  important  discoveries  the  true  relation  of 
the  lower  organisms  to  infectious  diseases  was  scientifi- 
cally pointed  out.  With  the  investigations  of  Pasteur 
upon  the  cause  of  putrefaction  in  beer  and  the  souring 
of  wine  ;  with  the  discovery  by  Pollender  and  Davaine 
of  the  presence  of  rod-shaped  organisms  in  the  blood  of 


22  BACTERIOLOGY. 

animals  dead  of  splenic  fever,  and  with  the  progress 
of  knowledge  upon  the  parasitic  nature  of  certain  dis- 
eases of  plants,  the  old  question  of  "  contagium  ani- 
matum  "  again  began  to  receive  attention.  It  was  taken 
up  by  Henle,  and  it  was  he  who  first  logically  taught 
this  doctrine  of  infection. 

The  main  point,  however,  that  had  occupied  the  atten- 
tion of  scientific  men  from  time  to  time  for  a  period  of 
about  two  hundred  years  subsequent  to  Leeuwenhoek's 
discoveries  was  the  origin  of  these  bodies.  Do  they 
generate  spontaneously,  or  are  they  the  descendants  of 
pre-existing  creatures  of  the  same  kind  ?  was  the  all- 
important  question.  Among  the  earlier  participants 
in  this  discussion  were  many  of  the  most  distinguished 
men  of  the  day. 

In  1749  Needham,  who  held  firmly  to  the  opinion 
that  the  bodies  which  were  attracting  such  general  atten- 
tion developed  spontaneously  as  the  result  of  vegetative 
changes  in  the  substances  in  which  they  were  found, 
attempted  to  demonstrate  by  experiment  the  grounds 
upon  which  he  held  this  view.  He  maintained  that 
the  bacteria  which  appeared  about  a  grain  of  barley 
germinating  in  a  carefully  covered  watch-crystal  of 
water  were  the  result  of  changes  going  on  in  the  barley- 
grain  itself,  incidental  to  its  germination. 

Spallanzani,  in  1769,  drew  attention  to  the  laxity  of 
the  methods  employed  by  Needham,  and  demonstrated 
that  if  infusions  of  decomposable  vegetable  matter  were 
placed  in  flasks,  which  were  then  hermetically  sealed, 
and  the  flasks  and  their  contents  allowed  to  remain 
for  a  time  in  a  vessel  of  boiling  water,  neither  could 
living  organisms  be  detected  nor  would  decomposition 
appear  in  the  infusions  so  treated.  The  objection  raised 


INTRODUCTION.  23 

by  Treviranus,  viz.,  that  the  high  temperature  to  which 
the  infusions  had  been  subjected  had  so  altered  them 
and  the  air  about  them  that  the  conditions  favorable  to 
spontaneous  generation  no  longer  existed,  was  promptly 
met  by  Spallanzani,  when  he  gently  tapped  one  of  the 
flasks  that  had  been  boiled  against  a  hard  object  until  a 
minute  crack  was  produced  ;  invariably  organisms  and 
decomposition  appeared  in  the  flask  thus  treated. 

From  the  time  of  the  experiments  of  Spallanzani 
until  as  late  as  1836  but  little  advance  was  made  in  the 
elucidation  of  this  obscure  problem. 

In  1836  Schulze  attracted  attention  to  the""  subject  by 
the  convincing  nature  of  his  investigations.  He  showed 
that  if  the  air  which  gained  access  to  boiled  infusions 
was  robbed  of  its  living  organisms  by  being  caused  to 
pass  through  strong  acid  or  alkaline  solutions  no  decom- 
position occurred,  and  living  organisms  could  not  be 
detected  in  the  infusions.  Following  quickly  upon 
this  contribution  came  Schwann,  in  1837,  and  somewhat 
later  (1854)  Schroder  and  Dusch,  with  similar  results 
obtained  by  somewhat  different  means.  Schwann  de- 
prived the  air  which  passed  to  his  infusions  of  its  living 
particles  by  conducting  it  through  highly  heated  tubes ; 
whereas  Schroder  and  Dusch,  by  means  of  cotton-wool 
interposed  between  the  boiled  infusions  and  the  outside 
air,  robbed  the  air  passing  to  the  infusions  of  its  organ- 
isms by  the  simple  process  of  filtration.  In  1860  Hoff- 
mann and  in  1861  Chevreul  and  Pasteur  demonstrated 
that  the  precautions  taken  by  preceding  investigators 
for  rendering  the  air  which  entered  these  flasks  free 
from  bacteria  were  not  necessary;  that  all  that  was 
necessary  to  prevent  the  access  of  bacteria  to  the  infu- 
sions in  the  flasks  was  to  draw  out  the  neck  of  the  flask 


24  BACTERIOLOGY. 

into  a  fine  tube,  bend  it  down  along  the  side  of  the 
flask,  and  then  bend  it  up  again  a  few  centimetres  from 
its  extremity,  and  leave  the  mouth  open.  The  infusion 
was  then  to  be  boiled  in  the  flask  thus  prepared  and  the 
mouth  of  the  tube  left  open.  The  organisms  which 
now  fell  into  the  open  end  of  the  tube  were  arrested  by 
the  drop  of  water  of  condensation  which  collected  at  its 
lowest  angle,  and  none  could  enter  the  flask. 

While  from  our  modern  standpoint  the  results  of 
these  investigations  seem  to  be  of  a  most  convincing 
nature,  yet  there  were  many  at  the  time  who  required 
additional  proof  that  " spontaneous  generation"  was 
not  the  explanation  for  the  mysterious  appearance 
of  these  minute  living  creatures.  The  majority,  if 
not  all,  of  such  doubts  were  subsequently  dissipated 
through  the  well-known  investigations  of  Tyndall  upon 
the  floating  matters  of  the  air.  In  these  studies  he 
demonstrated  by  numerous  ingenious  and  instructive 
experiments  that  the  presence  of  living  organisms  in 
decomposing  fluids  was  always  to  be  explained  either 
by  the  pre-existence  of  similar  living  forms  in  the  infu- 
sion or  upon  the  walls  of  the  vessel  containing  it,  or 
by  the  infusion  having  been  exposed  to  air  which  had 
not  been  deprived  of  its  viable  organisms. 

Throughout  all  the  work  bearing  upon  this  subject 
from  the  time  of  Spallanzani  to  that  of  Tyndall,  certain 
irregularities  were  constantly  appearing.  It  was  found 
that  particular  substances  required  to  be  heated  for  a 
much  longer  time  than  was  needed  to  render  other 
substances  free  from  living  organisms,  and  even  with 
the  most  careful  precautions  decomposition  would  occa- 
sionally occur. 

In  1762  Bonnet,  who  was  deeply  interested  in  this 


INTRODUCTION.  25 

subject,  suggested,  in  reference  to  the  results  obtained 
by  Needham,  the  possibility  of  the  existence  of  "  germs 
or  their  eggs,"  which  had  the  power  to  resist  the  tem- 
perature to  which  some  of  the  infusions  employed  in 
Xeedham's  experiments  had  been  subjected. 

More  than  a  hundred  years  after  Bonnet  had  made 
this  purely  speculative  suggestion  it  became  the  happy 
privilege  of  Ferdinand  Cohn,  of  Breslau,  to  demon- 
strate its  accuracy. 

Cohn  repeated  the  foregoing  experiments  with  like 
results.  He  concluded  that  the  irregularities  could 
only  be  due  to  either  the  existence  of  more  resistant 
species  of  bacteria  or  to  more  resistant  stages  into 
which  certain  bacteria  have  the  property  of  passing. 
He  demonstrated  that  certain  of  the  rod-shaped  organ- 
isms possess  the  power  of  passing  into  a  resting-  or 
spore-stage  in  the  course  of  their  life-cycle,  and  when 
in  this  stage  they  are  much  less  susceptible  to  the  dele- 
terious action  of  high  temperatures  than  when  they  are 
growing  as  normal  vegetative  forms.  With  the  discov- 
ery of  these  more  resistant  spores  the  doctrine  of  spon- 
taneous generation  received  its  death-blow.  It  was  no 
longer  difficult  to  explain  the  inconsistencies  in  the  re- 
sults of  former  investigations,  nor  was  it  any  longer  to  be 
doubted  that  putrefaction  and  fermentation  were  the  re- 
sult of  bacterial_life*"and  not  the  cause  of  it,  and'that  these 
bacteria  were  the  offspring  of  pre-existing  similar  forms. 
In  other  words,  the  law  of  Harvey,  Omne  vivum  ex  ovo, 
or  its  modification,  Omne  vivum  ex  vivo,  was  shown  to 
apply  not  only  to  the  more  highly  organized  members 
of  the  animal  and  vegetable  kingdoms,  but  to  the  most 
microscopic,  unicellular  creatures  as  well. 

The  establishment  of  this  point  served  as  an  impetus 


26  BACTERIOLOGY. 

to  further  investigations,  and  as  the  all-important  ques- 
tion was  that  concerning  the  relation  of  these  micro- 
scopic organisms  to  disease,  attention  naturally  turned 
into  this  channel  of  study.  Even  before  the  hypothesis 
of  spontaneous  generation  had  received  its  final  refutation 
a  number  of  observations  of  a  most  important  nature  had 
been  made  by  investigators  who  had  long  since  ceased  to 
consider  spontaneous  generation  as  a  tenable  explanation 
of  the  origin  of  the  microscopic  living  particles. 

In  the  main,  these  studies  had  been  conducted  upon 
wounds  and  the  infections  to  which  they  are  liable ;  in 
fact,  the  evolution  of  our  knowledge  of  bacteriology  to 
its  present  development  is  so  intimately  associated  with 
this  particular  line  of  investigation  that  a  few  historical 
facts  in  connection  with  it  may  not  be  without  interest. 

The  observations  of  Rindfleisch,  in  1866,  in  which 
he  describes  the  presence  of  small,  pin-head  points  in 
the  myocardium  and  general  musculature  of  individuals 
that  had  died  as  a  result  of  infected  wounds,  represent, 
probably,  the  first  reliable  contribution  to  this  subject. 
He  studied  the  tissue-changes  round  about  these  points 
up  to  the  stage  of  miliary  abscess-formation.  He  refers 
to  the  organisms  as  "  vibrios."  Almost  simultaneously 
von  Recklinghausen  and  Waldeyer  described  similar 
changes  that  they  had  observed  in  pyaemia  and,  occa- 
sionally, secondary  to  typhoid  fever.  Von  Reckling- 
hausen  believed  the  granules  seen  in  the  abscess-points 
to  be  micrococci  and  not  tissue-detritus,  and  gave  as 
the  reason  that  they' were  regular  in  size  and  shape,  and 
gave  specific  reactions  with  particular  staining-fluids. 
Birch-Hirschfeld  was  able  to  trace  bacteria  found  in 
the  blood  and  organs  to  the  wound  as  the  point  of  en- 
trance, and  believed  both  the  local  and  the  constitutional 


INTRODUCTION.  27 

condition  to  stand  in  direct  ratio  to  the  number  of  spher- 
ical bacteria  present  in  the  wound.  He  observed  also 
that  as  the  organisms  increased  in  number  they  could 
often  be  found  within  the  bodies  of  pus-corpuscles. 
His  studies  of  pyaemia  led  him  to  the  important  con- 
clusion that  in  this  condition  micro-organisms  were 
always  present  in  the  blood. 

Of  immense  importance  to  the  subject  were  the  in- 
vestigations of  Klebs,  made  at  the  Military  Hospital 
at  Carlsruhe  in  1870-71.  He  not  only  saw,  as  others 
before  him  had  seen,  that  bacteria  were  present  in  dis- 
eases following  infection  of  wounds,  but  described  the 
manner  in  which  the  organisms  had  gained  entrance 
from  the  point  of  injury  to  the  internal  organs  and 
blood.  He  expressed  the  opinion  that  the  spherical 
and  rod-shaped  bodies  which  he  saw  in  the  secretions  of 
wounds  were  closely  allied,  and  he  gave  to  them  the 
designation  "  microsporon  septicum."  He  believed  that 
the  organisms  gained  access  to  the  tissues  round  about 
the  point  of  injury  both  by  the  aid  of  the  wandering 
leucocytes  and  by  being  forced  through  the  connec- 
tive-tissue lymph-spaces  by  the  mechanical  pressure  of 
muscular  contraction. 

On  erysipelatous  inflammations  secondary  to  injury 
important  investigations  were  also  being  made,  Wilde, 
Orth,  von  Recklinghausen,  Lukomsky,  Billroth,  Ehr- 
lich,  Fehleisen,  and  others  agreeing  that  in  these  con- 
ditions micro-organisms  could  always  be  detected  in  the 
lymph-channels  of  the  subcutaneous  tissues ;  and  through 
the  work  of  Oertel,  Nassiloff,  Classen,  Letzerich,  Klebs, 
and  Eberth  the  constant  presence  of  bacteria  in  the 
diphtheritic  deposits  at  times  seen  on  open  wounds  was 
established. 


28  BACTERIOLOGY. 

Simple  and  natural  as  all  this  may  seem  to  us  now, 
the  stage  to  which  the  subject  had  developed  when  these 
observations  were  recorded  did  not  admit  of  their  meet- 
ing with  unconditional  acceptance.  The  only  strong 
argument  in  favor  of  the  etiological  relation  of  the 
organisms  that  had  been  seen  to  the  diseases  with  which 
they  were  associated  was  the  constancy  of  this  associa- 
tion. No  efforts  had  been  made  to  isolate  them,  and 
few  or  none  to  reproduce  the  pathological  conditions  by 
inoculation.  Moreover,  not  a  small  number  of  inves- 
tigators were  skeptical  as  to  the  importance  of  these 
observations ;  many  claimed  that  micro-organisms  were 
normally  present  in  the  blood  and  tissues  of  the  body  ; 
and  some  even  urged  that  the  organisms  seen  in  dis- 
eased conditions  were  the  result  rather  than  the  cause 
of  the  maladies.?  It  is  hardly  necessary  to  do  more 
than  say  that  both  of  these  views  were  purely  specula- 
tive, and  have  never  had  a  single  reliable  experimental 
argument  in  their  favor.  Billroth  and  Tiegel,  who  held 
to  the  former  opinion,  did  endeavor  to  prove  their  posi- 
tion through  experimental  means  ;  but  the  methods  em- 
ployed by  them  were  of  such  an  untrustworthy  nature 
that  the  fallacy  of  deductions  drawn  from  them  was 
very  quickly  demonstrated  by  subsequent  investigators. 
Their  method  for  demonstrating  the  presence  of  micro- 
organisms in  normal  tissues  was  to  remove  bits  of  organs 
from  the  healthy  animal  body  with  heated  instruments 
and  drop  them  into  hot  melted  paraffin,  they  holding  that 
all  living  organisms  on  the  surface  of  the  tissues  would  be 
destroyed  by  the  high  temperature,  and  that  if  decom- 
position should  subsequently  occur  it  would  prove  that 
it  was  the  result  of  the  growth  of  bacteria  in  the  depths 
of  the  tissues  to  which  the  heat  had  not  penetrated. 


INTRODUCTION.  29 

Decomposition  did  usually  set  in,  and  they  accepted 
this  as  proof  of  the  accuracy  of  their  view.  Attention 
was,  however,  shortly  called  to  the  fact  that  in  cooling 
there  was  contraction  of  the  paraffin,  resulting  usually 
in  the  production  of  small  rents  and  cracks  in  which 
dust,  and  bacteria  lodged  upon  it,  could  accumulate  and 
finally  gain  access  to  the  tissues,  with  the  occurrence  of 
decomposition  as  a  consequence.  Their  results  were 
thus  explained  after  a  manner  analogous  to  that  em- 
ployed by  Spallanzani,  in  1769,  in  demonstrating  to 
Treviranus  the  fallacy  of  the  opinion  held  by  him  and 
the  accuracy  of  his  own  views,  viz.,  that  it  was  always 
through  the  access  of  organisms  from  without  that  de- 
composition primarily  originated.  (See  page  23.) 

Under  careful  precautions,  against  which  no  objection 
could  be  raised,  the  experiments  of  Bill  roth  and  Tiegel 
were  repeated  by  Pasteur,  Burdon-Sanderson,  and  Klebs, 
but  with  failure  in  every  instance  to  demonstrate  the 
presence  of  bacteria  in  the  healthy  living  tissues. 

The  fundamental  researches  of  Koch  (1881)  upon 
pathogenic  bacteria  and  their  relation  to  the  infectious 
diseases  of  animals  differed  from  those  of  preceding 
investigators  in  many  important  respects.  The  scien- 
tific methods  of  analysis  with  which  each  and  every 
obscure  problem  was  met  as  it  arose  served  at  once  to 
distinguish  him  as  a  pioneer  in  this  hitherto  but  imper- 
fectly cultivated  domain.  The  outcome  of  these  inves- 
tigations was  the  establishment  of  a  foundation  upon 
which  the  bacteriology  of  the  future  was  to  rest.  He,  for 
the  first  time,  demonstrated  that  distinct  varieties  of  infec- 
tion, as  evidenced  by  anatomical  changes,  are  due  in  many 
cases  to  the  activities  of  specific  micro-organisms,  and 
that  by  proper  methods  it  is  possible  to  isolate  these 


30  BACTERIOLOGY. 

organisms  in  pure  culture,  to  cultivate  them  indefinitely, 
to  reproduce  the  conditions  by  inoculation  of  these  pure 
cultures  into  susceptible  animals,  and  to  continue  the 
disease  at  will  by  continuous  inoculation  from  an  in- 
fected to  a  healthy  animal.  By  the  methods  that  he 
employed  he  demonstrated  a  series  of  separate  and  dis- 
tinct diseases  that  can  be  produced  in  mice  and  rabbits 
by  the  injection  of  putrid  substances  into  their  tissues. 
The  disease  known  as  septicaemia  of  mice,  likewise  a  dis- 
ease characterized  by  progressive  abscess-formation,  and 
pyaemia  and  septicaBmia  of  rabbits,  were  among  the  affec- 
tions produced  by  him  in  this  way.  It  was  in  the  course 
of  this  work  that  the  Abbe  system  of  substage  condens- 
ing apparatus  was  first  used  in  bacteriology ;  that  the 
aniline  dyes  suggested  by  Weigert  were  brought  into 
general  use  ;  that  the  isolation  and  cultivation  of  bacteria 
in  pure  culture  on  solid  media  were  shown  to  be  possible ; 
and  that  animals  were  employed  as  a  means  of  obtain- 
ing from  mixtures  pure  cultures  of  pathogenic  bacteria. 
With  the  bounteous  harvest  of  original  and  important 
suggestions  that  was  reaped  from  Koch's  classical  series 
of  investigations  bacteriology  reached  an  epoch  in  its 
development,  and  at  this  period  modern  bacteriology 
may  justly  be  said  to  have  had  its  birth. 

NOTE. — I  have  presented  only  the  most  prominent 
investigations  that  will  serve  to  indicate  the  lines  along 
which  the  subject  has  developed.  For  a  more  detailed 
account  of  the  historical  development  of  the  work  the 
reader  is  referred  to  Loffler's  instructive  and  enter- 
taining Vwlesungen  uber  die  geschichtliche  Entwickeluny 
der  Lehre  von  den  Baderien,  upon  which  I  have  drawn 
largely  in  preparing  the  foregoing  sketch. 


• 


CHAPTER   I. 

Definition  of  bacteria — Their  place  in  nature — Differences  between 
parasites  and  saprophytes— Nutrition  of  bacteria— Products  of  bac- 
teria— Their  relation  to  oxygen — Influence  of  temperature  upon 
their  growth. 

BY  the  term  bacteria  is  understood  that  large  group 
of  minute  vegetable  organisms  the  individual  members 
of  which  multiply  by  a  process  of  transverse  division. 
They  are  spherical,  oval,  rod-like,  and  spiral  in  shape, 
and  are  commonly  devoid  of  chlorophyll.1  Owing  to 
the  absence  of  chlorophyll  from  their  composition  bac- 
teria are  forced  to  obtain  their  nutritive  materials  from 
organic  matters  as  such,  and  lead,  therefore,  either  a 
>a  prophetic2  or  parasitic3  existence. 

Their  life-processes  are  so  rapid,  complex,  and  ener- 
getic that  they  result  in  the  most  profound  alterations 
in  the  structure  and  composition  of  the  materials  in  and 
upon  which  they  are  developing. 

Decomposition,  putrefaction,  and  fermentation  result 

1  Chlorophyll  is  the  green  coloring-matter  possessed  by  the  higher 
plants  by  means  of  which  they  are  enabled  in  the  presence  of  sunlight 
to  decompose  carbonic  acid  (COa)  and  ammonia  (NHs)  into  their  ele- 
mentary constituents. 

-  A  saprophyte  is  an  organism  that  obtains  its  nutrition  from  dead 
organic  matter. 

3  A  parasite  lives  always  at  the  expense  of  some  other  living,  organic 
creature,  known  as  its  host,  and  in  the  strictest  sense  of  the  word  can- 
not develop  upon  dead  matter.  There  is,  however,  a  group  of  so-called 
"facultative"  saprophytes  and  parasites  which  possess  the  power  of 
accommodating  themselves  to  existing  surroundings — at  one  time  lead- 
ing a  parasitic,  at  another  time  a  saprophytic  existence. 

31 


32  BACTERIOLOGY. 

from  the  activities  of  the  saprophytic  bacteria ;  while  the 
changes  brought  about  in  the  tissues  of  their  host  by  the 
purely  parasitic  forms  find  expression  in  disease-proc- 
esses and  not  infrequently  in  complete  death. 

The  role  played  in  nature  by  the  saprophytic  bacteria  is 
a  very  important  one.  Through  their  functional  activities 
the  highly  complicated  tissues  of  dead  animals  and  vege- 
tables are  resolved  into  the  simpler  compounds,  carbonic 
acid,  water,  and  ammonia,  in  which  form  they  may  be 
taken  up  and  appropriated  as  nourishment  by  the  more 
highly  organized  members  of  the  vegetable  kingdom. 
It  is  through  this  ultimate  production  of  carbonic  acid, 
ammonia,  and  water  by  bacteria,  as  end-products  in 
the  processes  of  decomposition  and  fermentation  of  dead 
animal  and  vegetable  tissues,  that  the  demands  of  grow- 
ing vegetation  for  these  compounds  are  supplied. 

The  chlorophyll  plants  do  not  possess  the  power  of 
obtaining  their  carbon  and  nitrogen  from  such  highly 
organized  and  complicated  substances  as  serve  for  the 
nutrition  of  bacteria,  and  as  the  production  of  the  simpler 
compounds,  carbon  dioxide  and  ammonia,  by  the  animal 
world  is  not  sufficient  to  meet  the  demands  of  the  chlo- 
rophyll plants,  the  importance  of  the  part  played  by 
bacteria  in  making  up  this  deficit  cannot  be  overesti- 
mated. Were  it  not  for  the  activity  of  these  microscopic 
living  creatures  all  life  upon  the  surface  of  the  earth 
would  cease.  Deprive  higher  vegetation  of  the  carbon 
and  nitrogen  supplied  to  it  as  a  result  of  bacterial  ac- 
tivity, and  its  development  comes  rapidly  to  an  end ; 
rob  the  animal  kingdom  of  the  food-stuffs  supplied  to 
it  by  the  vegetable  world,  and  life  is  no  longer  pos- 
sible. It  is  plain,  therefore,  that  the  saprophytes,  which 
represent  the  large  majority  of  all  bacteria,  must  be 


THEIR  PLACE  IN  ^NATURE.  33 

looked  upon  in  the  light  of  benefactors,  without  which 
existence  would  be  impossible. 

With  the  parasites,  on  the  other  hand,  the  conditions 
are  far  from  analogous.  Through  their  metabolic  activ- 
ities there  is  constantly  a  loss,  rather  than  a  gain,  to  both 
the  animal  and  vegetable  kingdoms.  Their  host  must 
always  be  a  living  body  in  which  exist  conditions 
favorable  to  their  development,  and  from  which  they 
appropriate  substances  that  are  necessary  to  the  health 
and  life  of  the  organism  to  which  they  have  found 
access ;  at  the  same  time  they  eliminate  substances  as 
products  of  their  nutrition  that  are  directly  poisonous 
to  the  tissues  in  which  they  are  growing. 

In  their  relations  to  terrestrial  life,  the  positions  oc- 
cupied by  the  two  functionally  different  groups,  the  sap- 
rophytes on  the  one  hand,  and  the  parasites  on  the  other, 
are  diametrically  opposite  :  the  saprophytic  forms  stand  in 
the  relation  of  benefactors,  in  resolving  dead  animal  and 
vegetable  bodies  into  their  component  parts,  which  serve 
as  food  for  living  vegetation,  and  at  the  same  time  they 
remove  from  the  surface  of  the  earth  the  remains  of  all 
dead  organic  substances ;  while  the  parasitic  group  exists 
only  at  the  expense  of  the  more  highly  organized  mem- 
bers of  both  the  animal  and  vegetable  kingdoms.  It  is 
to  the  parasitic  group  that  the  pathogenic 1  organisms  / 
belong. 

In  addition  to  the  saprophytes  that  are  concerned 'in 
the  changes  to  which  allusion  has  just  been  made,  there 
exist  other  saprophytic  forms  whose  life-processes  result 
in  specific  changes  of  most  interesting  and  important 
natures.  Some  of  these  are  characterized  by  their  prop- 

1  Pathogenic  organisms  are  those  which  possess  the  property  of  pro- 
ducing disease. 
3 


34  BACTERIOLOGY. 

erty  of  producing  pigments  of  different  color  ;  these  are 
known  as  the  chromogenic1  forms.  Just  what  is  their 
exact  role  in  nature  it  is  difficult  to  say ;  but  it  is  prob- 
able that,  in  addition  to  their  most  conspicuous  function 
of  color-production,  they  are  also  in  some  way  concerned 
in  the  omnipresent  process  of  disintegration  which  is 
constantly  going  on  in  all  dead  organic  substances. 
From  the  investigations  of  Beyerinck  it  would  seem 
that  in  some  of  these  chromogenic  forms  the  pigment 
is  an  integral  part  of  the  bacteria  themselves ;  in  others 
that  it  is  an  excretory  product  of  species  that  are  them- 
selves colorless ;  while  in  still  others,  that  it  is  an 
excretory  product  which  remains  intimately  associated 
with  the  bacterial  cells  and  in  part  or  wholly  stains 
them. 

Others,  the  so-called  photogenic  or  phosphorescent 
bacteria,  possess  the  property  of  producing  light  or 
of  illuminating  the  medium  on  which  they  grow  by 
a  peculiar  phosphorescence.  These  are  found  in  sea- 
water  and  in  decomposing  phosphorescent  fish  and 
meat. 

Still  others,  the  so-called  zymogenic  bacteria,  are  con- 
cerned in  the  various  fermentations,  such,  for  instance, 
as  acetic-,  lactic-,  and  butyric-acid  fermentations  ;  and 
many  of  the  industries,  such,  for  example,  as  those 
concerned  in  the  making  of  wine,  beer,  cheese,  butter, 
and  indigo,  are  more  or  less  directly  dependent  upon 
the  fermentation  that  accompanies  the  growth  of  pecu- 
liar species  of  bacteria  in  those  materials. 

Other  species  of  bacteria  liberate  in  the  course  of 
their  growth  soluble  proteolytic  ferments  or  enzymes, 
more  or  less  analogous  to  pepsin  and  trypsin  in  their 

1  Chromogenic  :  possessing  the  property  of  producing  color. 


VARIETIES  OF  BACTERIA.  35 

action  upon  proteids  ;  while  others  generate  ferments 
analogous  to  diastase  and  to  invertin  :  the  former  con- 
verting starch  into  sugar,  and  the  latter  having  the 
power  of  converting  cane-  into  grape-sugar. 

The  mjorogenic  bacteria  are  those  that  produce  the 
particular  fermentation  that  we  know  as  putrefac- 
tion. 

Another  very  important  saprophytic  group  comprises 
the  so-called  nitrifying  and  denitrifying  bacteria,  whose 
activities  are  concerned  in  specific  forms  of  fermentation  : 
the  former  oxidizing  ammonia  to  nitrous  and  nitric  acids  ; 
the  latter  reducing  nitric  acid  to  nitrous  acid  and  am- 
monia. It  is  through  their  association  (symbiosis)  with 
the  nitrifying  bacteria  that  certain  plants,  the  legumi- 
nous, are  enabled  to  make  up  their  nitrogen  deficit  in 
part  from  the  free  nitrogen  of  the  air.  The  discovery 
of  this  phenomenon  gave  to  free  atmospheric  nitro- 
gen a  biological  significance  that  had  hitherto  been 
denied  it. 

The  so-called  thiogenic  bacteria  convert  sulphuretted 
hydrogen  into  higher  sulphur  compounds. 

The  progress  of  many  forms  of  fermentation  and 
decomposition  is  accompanied  by  more  or  less  elevation 
of  temperature.  This  is  seen  in  the  curing  of  manure, 
tobacco,  malt,  etc. 

As  stated,  bacteria  that  produce  disease  are  known 
as  jtftfhoyemc.'  They  induce  disease  by  their  poisonous 
action  upon  the  tissues  in  which  they  are  located.  The 
materials  of  which  certain  species  of  bacteria  are  con- 
structed, and  the  products  of  growth  of  certain  others, 
are  of  the  greatest  importance  in  their  relation  to  animal 
and  human  pathology.  Particular  species,  while  not  elim- 
inating soluble  poisons  as  a  product  of  metabolism,  are 


36  BACTERIOLOGY. 

nevertheless  themselves  built  up  of  poisonous  proteids,  or 
of  proteids  with  which  toxic  materials  are  so  intimately 
associated  that  they  can  only  be  isolated  by  the  most  re- 
fined and  elaborate  chemical  manipulations.  Others  pro- 
duce in  the  course  of  their  growth  soluble  poisons  that  can 
readily  be  separated  by  very  simple  methods  from  the  bac- 
teria that  produced  them.  The  proteid  matters  making 
up  the  bodies  of  many  species  of  bacteria,  even  those  not 
conspicuously  pathogenic,  have  been  shown  by  Buchner 
to  induce  disease  when  isolated  and  injected  into  the 
tissues  of  animals ;  in  some  cases  causing  only  rise  of 
body-temperature,  in  others  acute  inflammatory  proc- 
esses with  pus-formation.  To  such  proteids  Buchner 
has  given  the  name  bacterial  proteins. 

The  poisonous  soluble  products  of  bacterial  growth 
are  severally  known  as  toxins,  toxalbumins,  and  pto- 
mains :  toxins  being,  in  general,  uncrystallizable  poisons 
of  whose  intimate  chemical  nature  little  or  nothing  is 
known ;  toxalbumins  being  those  that  partake  of  the 
chemical  nature  of  albumins ;  while  ptomains  are  crys- 
tallizable  products  of  bacterial  activity  which,  physically 
speaking,  are  analogous  to  the  ordinary  vegetable  alka- 
loids. 

We  have  said  that  through  the  agency  of  chlorophyll, 
in  the  presence  of  sunlight,  the  green  plants  are  enabled 
to  obtain  the  amount  of  nitrogen  and  carbon  which  is 
necessary  to  their  growth  from  such  simple  bodies  as 
carbon  dioxide  and  ammonia,  which  they  decompose 
into  their  elementary  constituents.  The  bacteria,  on 
the  other  hand,  owing  to  the  absence  of  chlorophyll 
from -their  tissues,  do  not  possess  this  power.  They 
must,  therefore,  have  their  carbon  and  nitrogen  pre- 


NUTRITION  OF  BACTERIA.  37 

sented  as  such,  in  the  form  of  decomposable  organic 
substances. 

In  general,  bacteria  obtain  their  nitrogen  most 
readily  from  soluble  albumins,  and  to  a  certain  extent, 
but  by  no  means  so  easily,  from  salts  of  ammonium.  In 
some  of  Nageli's  experiments  it  appeared  probable  that 
they  could  obtain  the  necessary  amount  of  nitrogen 
from  inorganic  nitrates.  At  all  events,  he  was  able 
in  certain  cases  to  demonstrate  a  reduction  of  nitric  to 
nitrous  acid  and  ultimately  to  ammonia.  Neverthe- 
less, in  all  of  these  experiments  circumstances  point  to 
the  probability  that  the  nitrogen  obtained  by  the  bac- 
teria for  building  up  their  tissues  in  the  course  of 
their  development  was  derived  from  some  source 
other  than  the  nitric  acid  or  the  nitrates,  and  that  the 
reduction  of  this  acid  was  most  probably  a  secondary 
phenomenon.  It  must  be  borne  in  mind,  however,  that 
there  exists  a  specific  group  of  bacteria,  the  nitrifying 
bacteria,  that  apparently  increase  and  multiply  without 
appropriating  proteid  nutrition.  They  are  concerned 
in  the  particular  form  of  fermentation  that  results  in 
the  oxidation  of  ammonia  to  nitrous  and  nitric  acids,  a 
process  everywhere  in  progress  in  the  superficial  layers 
of  the  soil. 

For  the  supply  of  carbon  many  of  the  carbon  com- 
pounds serve  as  sources  upon  which  the  bacteria  can 
draw.  The  carbon  deficit,  for  example,  can  be  obtained 
from  sugar  and  bodies  of  like  composition ;  from  glyc- 
erin and  many  of  the  fatty  acids ;  and  from  the  alka- 
line salts  of  tartaric,  citric,  malic,  lactic,  and  acetic 
acids.  In  some  instances  carbon  compounds,  which 
when  present  in  concentrated  form  inhibit  the  growth 
of  bacteria,  may,  when  highly  diluted,  serve  as  nutri- 


38  BACTERIOLOGY. 

tion  for  them.  Salicylic  acid  and  ethyl  alcohol  are  of 
this  class. 

In  addition  to  carbon  and  nitrogen,  water  is  essential 
to  the  life  and  development  of  bacteria.  Without  it 
no  development  occurs,  and  in  many  cases  drying  the 
organisms  results  in  their  death.  Certain  species  and 
developmental  forms,  on  the  contrary,  though  incapable 
of  multiplying  when  in  the  dry  state,  may  be  com- 
pletely deprived  of  their  water  without  causing  them 
to  lose  the  power  of  reproduction  under  favorable  con- 
ditions. 

Closer  study  of  bacteria,  and  a  more  intimate  ac- 
quaintance with  their  nutritive  changes,  demonstrate 
an  appreciable  variability  in  the  character  of  the  sub- 
stances best  suited  for  the  nutrition  of  different  species ; 
one  requiring  a  tolerably  concentrated  form  of  nutri- 
tion, while  another  needs  but  a  very  limited  amount 
of  proteid  substance  for  its  development.  Certain 
of  them  bring  about  most  profound  alterations  in  the 
media  in  which  they  exifet,  while  others  produce  but 
little  apparent  change.  s  In  one  case  alterations  in  the 
reaction  of  the  media'  will  be  conspicuous,  while  in 
another  no  such  variation  can  be  detected.  With  the 
growth  of  some  forms  products  resulting  from  specific 
processes  of  fermentation  appear.  Other  varieties  pro- 
duce poisons  of  remarkable  degrees  of  toxicity,  while  the 
growth  of  others  may  be  accompanied  by  the  evolution 
of  compounds  characteristic  of  putrefaction. 

In  considering  the  normal  development  of  bacteria 
we  must  not  lose  sight  of  the  fact  that  this  is  influenced 
both  by  the  quality  and  the  quantity  of  the  nutritive  mate- 
rials to  which  they  have  access,  and  by  the  character 
of  the  products  that  accumulate  in  these  materials  as  a 


NUTRITION  OF  BACTERIA.  39 

result  of  their  vital  processes.  Nitrogen  and  carbon 
compounds  may  be  present  in  amount  and  kind  entirely 
suitable  to  normal  bacterial  growth,  and  yet  this  may 
be  checked,  after  a  comparatively  short  time,  by  the 
accumulated  products  of  bacterial  metabolism,  some  of 
which  possess  the  property  of  inhibiting  growth  and 
ultimately  of  even  destroying  the  bacteria.  The  most 
common  and  conspicuous  examples  of  the  inhibiting 
conditions  that  are  coincident  with  bacterial  growth 
are  alterations  in  the  chemical  reaction  of  the  matters 
in  which  the  bacteria  are  developing. 

Since  the  majority  of  them  grow  best  in  media  of  a 
neutral  or  very  slightly  alkaline  reaction,  any  exces- 
sive development  of  alkalinity  or  acidity,  as  a  result 
of  growth,  arrests  development,  and  no  evidence  of 
life  or  further  multiplication  can  be  detected  until  this 
deviation  from  the  neutral  reaction  has  been  corrected. 

Of  considerable  importance  and  interest  in  the  study 
of  the  nutritive  changes  of  bacteria  is  the  difference  in 
their  relation  to  oxygen.  For  certain  species  free  oxy- 
gen is  essential  to  the  proper  performance  of  their 
functions  ;  in  another  group  no  evidence  of  life  can  be 
detected  under  its  access ;  while  in  a  third  group  oxy- 
gen appears  to  play  but  an  unimportant  role,  for  de- 
velopment occurs  as  well  with  as  without  it.  It  was 
Pasteur  wTho  first  demonstrated  the  existence  of  partic- 
ular species  of  bacteria  which  not  only  grow  and  multi- 
ply and  perform  definite  physiological  functions  with- 
out the  aid  of  free  oxygen,  but  to  the  existence  of  which 
it  is  positively  harmful.  To  these  he  gave  the  name 
anaerobic  bacteria,  in  contradistinction  to  the  aerobic 
group,  for  the  proper  performance  of  whose  functions 


40  BACTERIOLOGY. 

oxygen  is  essential.  In  addition  to  these  there  is  a 
third  group,  for  the  maintenance  of  whose  existence  the 
absence  or  presence  of  oxygen  is  apparently  of  no 
moment — development  progresses  as  well  with  as  with- 
out it ;  the  members  of  this  group  comprise  the  class 
known  as  facultative  in  their  relation  to  this  gas.  It  is 
to  this  third  group,  the  facultative,  that  the  majority  of 
bacteria  belong.  Since  all  growing  bacteria,  anaerobic 
as  well  as  aerobic,  generate  carbonic  acid  in  the  course 
of  their  development,  it  is  evident  that  oxygen  must 
in  reality  be  obtained  by  them  from  some  source,  and 
must  be  regarded  as  essential  to  their  life-processes; 
but  the  manner  in  which  it  is  appropriated  by  them 
varies,  the  aerobic  species  taking  it  from  the  air  as  free 
oxygen,  while  the  anaerobic  species,  not  possessed  of 
this  power,  obtain  it  through  the  decomposition  of  more 
or  less  stable  oxygen-containing  compounds. 

Though  the  multiplication  of  the  facultative  varieties 
is  not  interfered  with  by  either  the  presence  or  absence 
of  free  oxygen,  yet  experiments  demonstrate  that  the 
products  of  their  growth  are  different  under  the  varying 
conditions  of  absence  or  presence  of  this  gas.  For  ex- 
ample :  in  the  case  of  certain  of  the  chromogenic  forms 
the  presence  or  absence  of  oxygen  has  a  very  decided 
effect  upon  the  production  of  the  pigments  by  which 
they  are  characterized. 

NOTE. — Observe  the  difference  between  the  intensity 
of  color  produced  upon  the  surface  of  the  medium  and 
that  along  the  track  of  the  needle  in  stab-cultures  of 
bacillus  prodigiosus  and  of  spirillum  rubrum.  In  the 
former  the  red  color  is  apparently  a  product  dependent 
upon  the  presence  of  oxygen,  while  in  the  latter  the 


GROWTH  AND  DEVELOPMENT  OF  BACTERIA.    41 

greatest  intensity  of  color  occurs  at  the  point  furthest 
removed  from  the  action  of  oxygen. 

Another  factor  which  plays  a  highly  important  part 
in  the  biological  functions  of  these  organisms  is  the 
temperature  under  which  they  exist.  The  extremes  of 
temperature  between  which  the  majority  of  bacteria  are 
known  to  grow  range  from  5.5°  to  43°  C.  At  the 
former  temperature  development  is  hardly  appreciable  ; 
it  becomes  more  and  more  active  until  38°  C.  is  reached, 
when  it  is  at  its  optimum,  and,  as  a  rule,  ceases  at 
43°  C. ;  though  species  exist  that  multiply  at  as  high 
a  temperature  as  70°  C.  and  others  at  as  low  as  0°  C. 
The  studies  of  Globig,1  Miquel,2  and  Macfadyen  and 
Bloxall 3  have  demonstrated  that  there  exist  in  the  soil, 
in  water,  in  faeces,  in  sewage,  in  dust,  and,  in  fact,  prac- 
tically everywhere,  bacteria  that  under  artificial  culti- 
vation show  no  evidence  of  life  at  a  temperature  lower 
than  60°  to  65°  C.,  and  will  even  grow  at  as  high 
a  temperature  as  70°  to  75°  C.,  degrees  of  heat  suffi- 
cient for  the  coagulation  of  albumin  !  Rabinowitsch 4 
has  likewise  described  a  number  of  species  of  these 
thermoph'dic  bacteria,  as  they  are  called ;  but  states  that 
it  \vas  possible  in  her  experiments  to  obtain  evidence 
of  their  growth  at  a  lower  temperature  (34°  to  44°  C.), 
as  well  as  at  the  higher  temperature  mentioned  by  pre- 
ceding investigators. 

The  most  favorable  temperature  for  the  development 
of  pathogenic  bacteria  is  that  of  the  human  body,  viz., 

1  Globig :  Zeitschrift  fur  Hygiene,  Bd.  iii.  S.  294. 

2  Miquel :  Annales  de  Mierographie,  1888,  pp.  4  to  10. 

3  Macfadyen  and  Bloxall :  Journal  of  Path,  and   Bact.,  vol.  iii.  part  i. 

4  Rabinowitsch :  Zeitschrift  fur  Hygiene  u.  Infectionskrankheiten, 
Bd.  xx.  Heft  1,  S.  154  to  164. 


42  BACTERIOLOGY. 

37.5°  C.  There  are  a  number  of  bacteria  commonly 
present  in  water,  the  so-called  normal  water  bacteria, 
that  grow  best  at  about  20°  C. 

Under  natural  conditions  it  frequently  occurs  that 
the  development  of  one  species  or  group  of  species  of 
bacteria  is  directly  dependent  upon  the  functional  ac- 
tivities of  another  totally  distinct  species,  the  growth  of 
one  group  resulting  in  conditions  that  are  of  vital  im- 
portance to  the  existence  of  the  other.  This  interde- 
pendence of  species  is  known  as  symbiosis.  It  is  observed, 
for  instance,  in  the  course  of  putrefaction,  where, 
through  exhaustion  of  free  oxygen  by  the  actively 
germinating  aerobic  varieties,  the  conditions  are  sup- 
plied that  enable  the  anaerobic  species  to  develop  and 
exercise  their  biological  activities.  Again,  through  the 
proteolytic  activity  of  enzymes  produced  by  certain 
species  of  bacteria,  other  species  are  supplied  with  nu- 
trition that  would  otherwise  be  unassimilable  or  only 
imperfectly  so.  Similar  symbiotic  relations  between 
bacteria  and  higher  plants  are  also  noticed,  notably  that 
between  certain  bacteria  of  the  soil  and  the  group  of 
leguminous  plants,  whereby  the  latter  are  enabled, 
through  the  assistance  of  the  former,  to  make  up  their 
nitrogen  deficit  in  large  part  from  the  free  nitrogen  of 
the  atmosphere.  (See  page  35.) 

Another  interesting  biological  peculiarity  of  bacteria 
is  that  discovered  by  Pfeffer,  known  as  ^hemotaxis^  This 
term  indicates  peculiar  phenomena  of  attraction  and  of 
repulsion  that  are  exhibited  by  motile  bacteria  when  in 
the  presence  of  solutions  of  bodies  of  various  chemi- 
cal composition.  Thus,  for  instance,  when  a  neutral 
fluid  (a  drop  of  water)  containing  motile  bacteria  is 
brought  in  contact  with  a  weak  solution  of  either  pep- 


GROWTH  AND  DEVELOPMENT  OF  BACTERIA.    43 

tone,  sodium  chloride,  or  dextrin,  the  bacteria  are  at 
once  attracted  toward  the  solution  ;  this  phenomenon  is 
designated  "  positive  chemotaxis."  On  the  other  hand, 
if  brought  in  contact  with  an  acid,  an  alkaline,  or  an 
alcoholic  solution,  the  bacteria  are  repelled  or  driven 
from  the  point  at  which  the  two  fluids  are  diffusing ; 
this  phenomenon  is  designated  "  negative  chemotaxis." 
In  this  connection  it  is  important  to  note  that  the  wan- 
dering cells  of  the  animal  body,  the  leucocytes,  exhibit 
also  these  chemotactic  phenomena ;  and  it  is  especially 
necessary  to  a  complete  comprehension  of  the  process 
of  suppuration  to  bear  in  mind  that  among  the  sub- 
stances which  have  the  greatest  attraction  for  these 
wandering  cells,  are  the  products  of  growth  of  certain 
bacteria  in  some  cases,  and  the  protoplasmic  constit- 
uents of  the  bacteria  themselves  in  others. 

From  what  has  been  learned,  it  may  be  said,  in 
general,  that  for  the  growth  and  development  of  bacteria 
organic  matter  of  a  neutral  or  slightly  alkaline  reaction, 
in  the  presence  of  moisture  and  at  a  suitable  tempera- 
ture, is  necessary.  From  this  can  be  formed  some  idea 
of  the  omnipresence  in  nature  of  these  minute  vegetable 
forms.  Wherever  these  conditions  obtain  bacteria  may 
be  found. 


CHAPTER    II. 

Morphology l  of  bacteria — Grouping — Mode  of  Multiplication — Spore- 
formation — Motili  ty. 

IN  structure  the  bacteria  are  unicellular,  always  de- 
veloping from  pre-existing  cells  of  the  same  character 
and  never  appearing  spontaneously.  They  are  seen  to 
occur  as  spherical,  rod-  and  spiral-shaped  bodies  that 
multiply  by  the  simple  process  of  transverse  division, 
belonging,  therefore,  to  the  schizomycetes  or  fission 
fungi. 

The  classifications  of  the  older  authors  and  of  the 
botanists  being  usually  upon  purely  morphological  pecu- 
liarities, and,  as  a  rule,  taking  into  consideration  all  the 
slight  variations  that  are  seen  to  occur  in  the  size  and 
shape  of  one  and  the  same  species,  and  of  closely  allied 
species,  are  more  or  less  complicated.  The  present 
tendency  is  to  simplify  this  morphological  classification, 
and  to  distribute  the  bacteria  into  three  great  groups, 
with  their  subdivisions,  the  members  of  each  group 
being  identified  by  their  individual  outline,  viz.,  that 
of  a  sphere,  a  rod,  or  a  spiral.  To  these  three  grand 
divisions  are  given  the  names  cocci  or  micrococci,  bacilli, 
and  spirilla. 

In  the  group  micrococci  belong  all  spherical  forms— 
i.  6.,  all  those  forms  the  isolated  individual  members 
of  which  are  practically  of  the  same  diameter  in  all 
directions.  (See  Fig.  1,  a,  b,  c,  rf,  e.) 

1  Morphology  :  pertaining  to  shape,  outline,  structure. 
44 


Q&OUPtNQ. 


45 


The  bacilli  comprise  all  oval  or  rod-formed  bacteria. 
(See  Fig.  2.) 


FIG.  1. 


%£*     <$° 

Cx 

c 


o.". 


c  a  e 

a.  Staphylococci.    b.  Streptococci,    c.  Diplococci.     d.  Tetrads,     e.  Sarcinse. 

FIG.  2. 


rt.  Pacilli  in  pairs.    6.  Single  bacilli,    cand  d.  Bacilli  in  threads. 
e  and  /.  Bacilli  of  variable  morphology. 

FIG.  3. 


*(  6  C  r/ 

a  and  cf .  Spirilla  in  short  segments  and  longer  threads— the  so-called  comma 
forms  and  spirals,  b.  The  forms  known  as  spirochseta.  c.  The  thick  spirals 
sometimes  known  as  vibrios. 


46  BACTERIOLOGY. 

To  the  spirilla  belong  all  organisms  that  are  curved 
when  seen  in  short  segments,  or  when  in  longer  threads 
are  twisted  in  the  form  of  a  corkscrew.  (See  Fig.  3.) 

The  micrococci  are  subdivided  according  to  their  pre- 
vailing mode  of  grouping,  as  seen  in  growing  cultures, 
into  staphyloGQcci  —  those  growing  in  masses  like  clusters 
of  grapes  (see  Fig.  1,  a)  ;  streptococci  —  those  growing  in 
chains  consisting  of  a  number  of  individuals  strung 
together  like  beads  upon  a  string  (see  Fig.  1,  b)  ;  diplo- 
cocci  —  those  growing  in  pairs  (Fig.  1,  c)  ;  tetrads  —  those 
developing  as  fours  (Fig.  1,  d)  ;  and  sarcince  —  those 
dividing  into  fours,  eights,  etc.,  as  cubes  —  that  is,  in 
contradistinction  to  all  other  forms,  the  segmentation, 
which  is  rarely  complete,  takes  place  regularly  in  three 
directions  of  space,  so  that  when  growing  the  bundle  of 
segmenting  cells  presents  somewhat  the  appearance  of  a 
bale  of  cotton  (Fig.  1,  e). 

To  the  bacilli  belong  all  straight,  rod-shaped  bacteria 
—  i.  e.}  those  in  which  one  diameter  is  always  greater 
than  the  other. 

FIG.  4. 


a.  Bacillus  subtilis  with  spores.  &.  Bacillus  anthracis  with  spores,  c.  Clos- 
tridium  form  with  spores,    d.  Bacillus  of  tetanus  with  end  spores. 

In  this  group  are  found  those  organisms  the  life- 
cycle  of  many  of  which  presents  deviations  from  the 
simple  rod-shape.  Many  of  them  in  the  course  of 
development  increase  in  length  into  long  threads, 
along  which  traces  of  segmentation  may  usually  be 


GROUPING.  47 

found — the  anthrax  bacillus  and  bacillus  subtilis  are 
conspicuous  examples  of  this.  Again,  under  certain 
conditions,  many  of  them  possess  the  property  of  form- 
ing within  the  body  of  the  rods  oval,  glistening  spores 
(see  Fig.  4),  and,  if  the  conditions  are  not  altered,  the 
rods  may  entirely  disappear  and  nothing  be  left  in 
the  culture  but  these  oval  spores.  In  some  of  them 
this  phenomenon  of  spore-formation  is  accompanied  by 
an  enlargement  or  swelling  of  the  bacillus  at  the  point 
at  which  the  spore  is  located  (see  Fig.  4,  c  and  d). 
Again,  many  of  them,  from  unfavorable  conditions  of 
nutrition,  aeration,  or  temperature,  undergo  pathological 
changes — that  is,  the  individuals  themselves  experience 
degeneration  of  their  protoplasm  with  coincident  dis- 
tortion of  their  outline ;  they  are  thenoisually  referred 
to  as  "involution-forms"  (see  Fig.  5,  a  and  6).  In 

FIG.  5. 


a.  Spirillum  of  Asiatic  cholera  (comma  bacillus).    Involution-forms  of 
this  organism  as  seen  in  old  cultures. 

all  of  these  conditions,  however,  so  long  as  death  has 
not  occurred,  it  is  possible  to  cause  these  forms  to 
revert  to  the  typical  rods  from  which  they  originated, 
by  the  renewal  of  the  conditions  favorable  to  their  nor- 
mal vegetation. 

It  must  be  borne  in  mind,  though,  that  it  is  never 
possible  by  any  means  to  bring  about  changes  in  these 
organisms  that  will  result  in  the  permanent  conversion 
of  the  morphology  of  the  members  of  one  group  into 


48  BACTERIOLOGY. 

that  of  another — that  is,  one  can  never  produce  bacilli 
from  micrococci,  nor  vice  versa  ;  and  any  evidence  which 
may  be  presented  to  the  contrary  is  based  upon  untrust- 
worthy methods  of  observation. 

Not  infrequently  bacteria  may  be  observed  irregularly 
massed  together  as  a  pellicle.  When  in  this  condition 
they  are  held  together  by  a  gelatinous  material,  and  are 
known  as  zobgloea  of  bacteria.  (See  Fig.  6.) 

FIG.  6. 


.• 


Zooglcea  of  bacilli. 

Very  short  oval  bacilli  may  sometimes  be  mistaken 
for  micrococci,  and  at  times  micrococci  in  the  stage  of 
segmentation  into  diplococci  may  be  mistaken  for  short 
bacilli ;  but  by  careful  inspection  it  will  always  be 
possible  to  detect  a  continuous  outline  along  the  sides 
of  the  former,  and  a  slight  transverse  indentation  or 
partition-formation  between  the  segments  of  the  latter. 
The  high  index  of  refraction  of  spores,  the  property 
which  gives  to  them  their  glistening  appearance,  will 
always  serve  to  distinguish  them  from  micrococci.  This 
difference  in  refraction  is  especially  noticeable  if  the  illu- 
mination of  the  microscope  be  reduced  to  the  smallest 
possible  bundle  of  light-rays.  The  spores,  moreover, 
take  up  staining-reagents  much  less  readily  than  do 
the  micrococci.  The  most  reliable  differential  points, 
however,  are  the  infallible  properties  possessed  by  the 
spores  of  developing  into  bacilli,  and  by  the  spherical 


GERMINATION.  49 

organism  with  which  they  may  have  been  confounded 
of  always  producing  other  micrococci  of  the  same  round 
form. 

A  convenient  classification  of  bacilli  is  that  based 
upon  constant  characteristics  which  are  seen  to  ap- 
pear in  the  course  of  their  development  under  spe- 
cial conditions — certain  of  them  possessing  the  power 
of  forming  spores,  while  from  others  this  peculiarity 
is  absent. 

We  have  less  knowledge  of  the  life-history  of  the 
spiral  forms.  Efforts  toward  their  cultivation  under 
artificial  conditions  have  thus  far  been  successful  in 
only  a  comparatively  limited  number  of  cases.  Mor- 
phologically, they  are  thread-  or  rod-like  bodies  which 
are  twisted  into  the  form  of  spirals.  In  some  of  them 
the  turns  of  the  spiral  are  long,  in  others  quite  short. 
They  are  motile  and  multiply  apparently  by  the  simple 
process  of  fission.1  In  most  respects,  save  form  and 
the  property  of  producing  spores,  they  are  analogous  in 
their  mode  of  growth  to  the  bacilli. 

The  micrococci  multiply  by  simple  fission.  When 
development  is  in  progress  a  single  cell  will  be  seen  to 
elongate  slightly  in  one  of  its  diameters.  Over  the 
centre  of  the  long  axis  thus  formed  will  appear  a  slight 
indentation  in  the  outer  envelope  of  the  cell ;  this  inden- 
tation will  increase  in  extent  until  there  exist  eventually 
two  individuals  which  are  distinctly  spherical,  as  was 
the  parent  from  which  they  sprang,  or  they  will  remain 
together  for  a  time  as  diplococci ;  the  surfaces  now  in 
juxtaposition  are  flattened  against  one  another,  and  not 
infrequently  a  fine,  pale  dividing-line  may  be  seen 

1  Dividing  into  two  transversely. 


50  BACTERIOLOGY. 

between  the  two  cells.  (See  Fig.  1,  c  and  d.)  A  similar 
division  in  the  other  direction  will  now  result  in  the 
formation  of  groups  of  fours  as  tetrads. 

In  the  formation  of  staphylococci  such  division  occurs 
irregularly  in  all  directions,  resulting  in  the  production 
of  the  clusters  in  which  these  organisms  are  commonly 
seen.  (See  Fig.  1,  a.)  With  the  streptococci,  however, 
the  tendency  is  for  the  segmentation  to  continue  in  one 
direction  only,  resulting  in  the  production  of  long  chains 
of  4,  8,  and  12  individuals.  (See  Fig.  1,  6.) 

The  sarcinse  divide  more  or  less  regularly  in  three 
directions  of  space ;  but  instead  of  becoming  separated 
the  one  from  the  other  as  single  cells,  the  tendency  is 
for  the  segmentation  to  be  incomplete,  the  cells  remain- 
ing together  in  masses.  The  indentations  upon  these 
masses  or  cubes,  which  indicate  the  point  of  incomplete 
fission,  give  to  these  bundles  of  cells  the  appearance 
commonly  ascribed  to  them — that  of  a  bale  of  cotton 
or  a  packet  of  rags.  (See  Fig.  1,  e.) 

The  multiplication  of  bacilli  is  in  the  main  similar  to 
that  given  for  the  micrococci.  A  dividing  cell  will  elon- 
gate slightly  in  the  direction  of  its  long  axis ;  an  inden- 
tation will  appear  about  midway  between  its  poles,  and 
will  become  deeper  and  deeper  until  eventually  two 
daughter-cells  will  be  formed.  This  process  may  occur 
in  such  a  way  that  the  two  young  bacilli  will  adhere 
together  by  their  adjacent  ends  in  much  the  same  way 
that  sausages  are  seen  to  be  held  together  in  strings 
(Fig.  2,/),  or  the  segmentation  may  take  place  more 
at  right  angles  to  the  long  axis,  so  that  the  proximal 
ends  of  the  young  cells  are  flattened,  while  the  distal 
extremities  may  be  rounded  or  slightly  pointed  (Fig. 
2,  e).  The  segmentation  of  the  anthrax  bacillus,  with 


SPORE-FORMATION.  51 

which  we  are  to  become  acquainted  later,  results,  when 
completed,  in  an  indentation  of  the  adjacent  extrem- 
ities of  the  young  segments,  so  that  by  the  aid  of 
high  magnifying  powers  these  surfaces  are  seen  to  be 
actually  concave.  Bacilli  never  divide  longitudinally. 

With  the  spore-forming  bacilli,  under  favorable  con- 
ditions of  nutrition  and  temperature,  the  same  is  seen 
to  occur  during  vegetation  ;  but  as  soon  as  these  condi- 
tions become  altered  by  the  exhaustion  of  nourishment, 
the  presence  of  detrimental  substances,  unfavorable 
temperatures,  etc.,  there  appears  in  their  life-cycle  the 
stage  to  which  we  have  referred  as  spore-formation. 
This  is  the  process  by  which  the  organisms  are  enabled 
to  enter  a  state  in  which  they  resist  deleterious  influ- 
ences to  a  much  higher  degree  than  is  possible  for  them 
when  in  the  growing  or  vegetative  condition. 

In  the  spore,  resting,  or  permanent  state,  as  it  is  vari- 
ously called,  no  evidence  of  life  whatever  is  given  by  the 
spores ;  though  as  soon  as  the  conditions  which  favor 
their  germination  have  been  renewed  these  spores  de- 
velop again  into  the  same  kind  of  cells  as  those  from 
which  they  originated,  and  the  appearances  observed  in 
the  vegetative  or  growing  stage  of  their  history  are 
repeated. 

Multiplication  of  spores,  as  such,  does  not  occur ;  they 
possess  the  power  of  developing  into  individual  rods  of 
the  same  nature  as  those  from  which  they  were  formed, 
but  not  of  f/ii'inr/  rise  to  a  direct  reproduction  of  spores. 

When  the  conditions  which  favor  spore-formation 
present,  the  protoplasm  of  the  vegetative  cells  is  seen 
to  undergo  a  change.  It  loses  its  normal  homogeneous 
appearance  and  becomes  marked  by  granular,  refractive 
points  of  irregular  shape  and  size.  These  eventually 


52  BACTERIOLOGY. 

coalesce,  leaving  the  remainder  of  the  cell  clear  and 
transparent.  When  this  coalescence  of  highly  refrac- 
tive particles  is  complete  the  spore  is  perfected.  In 
appearance  the  spore  is  oval  or  round,  and  very  highly 
refractive — glistening.  It  is  easily  differentiated  from 
the  remainder  of  the  cell,  which  now  consists  only 
of  a  cell-membrane  and  a  transparent,  clear  space 
which  surrounds  the  spore.  Eventually  both  the  cell- 
membrane  and  its  fluid  contents  disappear,  leaving  the 
oval  spore  free ;  it  then  gives  the  impression  of  being 
surrounded  by  a  dark,  sharply  defined  border.  It 
evinces  no  motion  other  than  the  mechanical  tremor 
common  to  all  insoluble  microscopic  particles  suspended 
in  fluids,  and  it  remains  quiescent  until  there  appear  con- 
ditions favorable  to  its  subsequent  development  into  a 
vegetative  form  similar  to  that  from  which  it  originated. 
Occasionally  the  membrane  of  the  vegetative  cell  in 
which  the  spore  is  formed  does  not  disappear  from 
around  it,  and  the  spore  may  then  be  seen  lying  in  a 
very  delicate  tubular  envelope.  Now  and  then,  rem- 
nants of  the  envelope  may  be  noticed  adhering  to  a 
spore  which  has  not  yet  become  completely  free. 

By  the  ordinary  methods  of  staining ;  spores  do  not 
become  colored,  so  that  they  appear  in  the  stained 
cells  as  pale,  transparent,  oval  bodies,  surrounded  by 
the  remainder  of  the  cell,  which  has  taken  up  the  dye. 

A  single  cell  produces  but  one  spore.  This  may  be 
located  either  at  an  extremity  or  in  the  centre  of  the 
cell.  (Fig.  4.) 

Occasionally  spore-formation  is  accompanied  by  an 
enlargement  of  the  cell  at  the  point  at  which  the  proc- 
ess is  in  progress.  As  a  result,  the  outline  of  the  cell 
loses  its  regular  rod  shape  and  becomes  that  of  a  club, 


MOTILITY. 


53 


a  drum-stick,  or  a  lozenge,  depending  upon  whether 
the  location  of  the  spore  is  to  be  at  the  pole  or  in  the 
centre  of  the  cell.  (See  Fig.  4,  e  and  d.) 

In  addition  to  the  property  of  spore-formation  there 
is  another  striking  difference  between  various  species 
of  the  rod-shaped  organisms,  namely,  the  property 
of  motility,  by  which  some  of  them  are  distinguished. 
This  power  of  motion  is  due  to  very  delicate,  hair- 


FIG.  7. 


a  b  c 

a.  Spiral  forms  with  a  flagellum  at  only  one  end.  6.  Bacillus  of  typhoid 
fever  with  flagella  given  off  from  all  sides,  c.  Large  spirals  from  stagnant 
water  with  wisps  of  flagella  at  their  ends  (spirillum  undula). 


like  appendages  or  flagella,  by  the  lashing  motions  of 
which  the  rods  possessing  them  are  propelled  through 
the  fluid.  In  some  cases  the  flagella  are  located  at 
but  one  end  of  a  bacillus,  either  singly  or  in  a  bunch ; 
again,  they  may  be  seen  at  both  poles ;  and  in  some 
cases,  especially  with  the  bacillus  of  typhoid  fever,  they 
are  given  off  from  the  whole  surface  of  the  rod.  (See 
Fig.  7.)  In  a  few  instances  similar  locomotive  organs 
have  been  detected  on  spherical  bacteria — i.  e.,  motile 
micrococci  have  been  observed. 

For  a  long  time  this  property  of  independent  motion 
was  assumed  to  be  due  to  the  possession  of  some  such 
form  of  locomotive  apparatus,  because  similar  appendages 


54  BACTERIOLOGY. 

had  been  seen  upon  some  of  the  large,  motile  spirilla  found 
in  stagnant  water,  but  it  was  not  until  recently  that  the 
accuracy  of  this  assumption  was  actually  demonstrated. 
By  a  special  method  of  staining  Lbffler  has  been  able, 
in  a  number  of  cases,  to  render  visible  these  hair-like 
appendages.  His  method  consists  in  the  employment 
of  a  mordant,  by  the  aid  of  which  the  flagella  are  caused 
to  retain  the  staining  and  thus  become  visible.  Lof- 
fler's  method  of  staining  will  be  found  in  the  chapter 
devoted  to  this  part  of  the  technique. 


CHAPTER    III. 

Principles  of  sterilization  by  heat — Methods  employed — Discontinued 
sterilization — Sterilization  under  pressure — Apparatus  employed — 
Chemical  disinfection  and  sterilization. 

OF  fundamental  importance  to  successful  bacterio- 
logical manipulations  are  acquaintance  with  the  prin- 
ciples underlying  the  methods  of  sterilization  and  dis- 
infection, and  familiarity  with  the  approved  methods 
of  applying  these  principles  in  practice. 

In  many  laboratories  it  is  customary  to  employ  the 
term  sterilization  for  the  destruction  of  bacteria  by  heat, 
and  the  term  disinfection  for  the  accomplishment  of  the 
same  end  through  the  use  of  chemical  agents.  This 
distinction  in  the  use  of  the  terms  is  not  strictly  correct, 
as  we  shall  endeavor  to  explain. 

The  laboratory  application  of  the  word  sterilization  for 
the  destruction  of  bacteria  by  high  temperatures  prob- 
ably arose  from  the  circumstance  that  culture-media, 
and  certain  other  articles  that  it  is  desirable  to  ren- 
der absolutely  free  from  bacterial  life,  are  not  treated 
by  chemical  agents  for  this  purpose,  but  are  exposed  to 
the  influence  of  heat  in  various  forms  of  apparatus 
known  as  sterilizers ;  and  the  process  is,  therefore, 
known  as  sterilization.  On  the  other  hand,  cultures 
no  longer  useful,  bits  of  infected  tissue,  and  apparatus 
generally  that  it  is  desirable  to  render  free  from  danger, 
are  commonly  subjected  for  a  time  to  the  action  of  chem- 
ical compounds  possessing  germicidal  properties — i.  e., 

55 


56  BACTERIOLOGY. 

to  the  action  of  disinfectants ;  and  the  process  is,  there- 
fore, known  as  disinfection,  though  the  same  end  can 
also  be  reached  by  the  application  of  heat  to  these  arti- 
cles. Strictly  speaking,  sterilization  implies  the  com- 
plete destruction  of  the  vitality  of  all  micro-organisms 
that  may  be  present  in  or  upon  the  substance  to  be 
sterilized,  and  can  be  accomplished  by  the  proper  appli- 
cation of  both  thermal  and  chemical  agents ;  while 
disinfection,  though  it  may,  need  not  of  necessity,  in- 
sure the  destruction  of  all  living  forms  that  are  present, 
but  only  of  those  possessing  the  power  of  infecting;  it 
may  or  may  not,  therefore,  be  complete  in  the  sense  of 
sterilization.  From  this  we  see  it  is  possible  to  accom- 
plish both  sterilization  and  disinfection  as  well  by  chemi- 
cal as  by  thermal  means. 

In  practice  the  employment  of  these  means  is  gov- 
erned by  circumstances.  In  the  laboratory  it  is  essen- 
tial that  all  culture-media  with  which  work  is  to  be 
conducted  should  be  free  from  living  bacteria  or  their 
spores — they  must  be  sterile ;  and  it  is  equally  impor- 
tant that  their  original  chemical  composition  should 
remain  unchanged.  It  is  evident,  therefore,  that  ster- 
ilization of  these  substances  by  means  of  chemicals  is 
out  of  the  question,  for,  while  the  media  could  be  thus 
sterilized,  it  would  be  necessary,  in  order  to  accomplish 
this,  to  add  to  them  substances  capable  not  only  of  de- 
stroying all  micro-organisms  present,  but  whose  pres- 
ence would  at  the  same  time  prevent  the  growth  of 
bacteria  that  are  to  be  subsequently  cultivated  in  these 
media — that  is  to  say,  after  performing  their  sterilizing 
or  germicidal  function  the  chemical  disinfectants  would, 
by  their  further  presence,  exhibit  their  antiseptic  prop- 
erties and  thus  render  the  material  useless  as  a  culture- 


STERILIZATION  BY  HEAT.  57 

medium.  Exceptions  to  this  are  seen,  however,  in  the 
case  of  certain  volatile  substances  possessing  disinfect- 
ant powers — chloroform  and  ether,  for  instance ;  these 
bodies,  after  exhibiting  their  germicidal  activities, 
may  be  driven  off  by  gentle  heat,  leaving  the  media 
quite  suitable  for  purposes  of  cultivation.  They  are 
not,  however,  in  general  use  in  this  capacity. 

The  circumstances  under  which  chemical  sterilization 
or  disinfection  is  practised  in  the  laboratory  are,  ordi- 
narily, either  those  in  which  it  is  desirable  to  render 
materials  free  from  danger  that  are  not  affected  by  the 
chemical  action  of  the  agents  used,  such  as  glass  appa- 
ratus, etc.,  or  where  destructive  changes  in  the  compo- 
sition of  the  substances  to  be  treated,  as  in  the  case 
of  old  cultures,  infected  tissues,  pathological  exudates, 
faeces,  etc.,  are  a  matter  of  no  consequence.  On  the 
other  hand,  for  the  sterilization  of  all  materials  to  be 
used  as  culture-media  heat  only  is  employed.1 

The  two  processes  will  be  explained  in  this  chapter, 
beginning  with 


STERILIZATION    BY    HEAT. 

Sterilization  by  means  of  high  temperature  is  accom- 
plished in  several  ways,  viz.,  by  subjecting  the  articles 
to  be  treated  to  a  high  temperature  in  a  properly  con- 
structed oven — this  is  known  as  dry  sterilization ;  by 
subjecting  them  to  the  action  of  streaming  or  live  steam 
at  the  temperature  of  100°  C. ;  and  by  subjecting  them 
to  the  action  of  steam  under  pressure,  under  which 

1  An  occasional  exception  to  this  is  the  use  of  chloroform,  mentioned 
above. 


58  BACTERIOLOGY. 

circumstance  the  temperature  to  which  they  are  ex- 
posed becomes  more  and  more  elevated  as  the  pressure 
increases. 

Experience  has  taught  us  that  the  process  of  ster- 
ilization by  dry  heat  is  of  limited  application  because 
of  its  many  disadvantages.  For  successful  sterilization 
by  the  method  of  dry  heat  not  only  is  a  relatively 
high  temperature  essential,  but  the  substances  under 
treatment  must  be  exposed  to  this  temperature  for  a 
comparatively  long  time.  The  penetration  of  dry  heat 
into  materials  which  are  to  be  sterilized  is,  moreover, 
much  less  thorough  than  that  of  steam.  Many  sub- 
stances of  vegetable  and  animal  origin  are  rendered 
valueless  by  subjection  to  the  dry  method  of  sterilization. 
For  these  reasons  comparatively  few  materials  can  be 
sterilized  in  this  way  without  seriously  impairing  their 
further  usefulness. 

Successful  sterilization  by  dry  heat  cannot  usually 
be  accomplished  at  a  temperature  lower  than  150°  C., 
and  to  this  degree  of  heat  the  objects  should  be  sub- 
jected for  not  less  than  one  hour.  For  the  sterilization, 
therefore,  of  the  organic  materials  of  which  the  media 
employed  in  bacteriological  work  are  composed,  ancl  of 
domestic  articles,  such  as  cotton,  woollen,  wooden,  and 
leather  articles,  this  method  is  wholly  unsuitable.  In 
bacteriological  work  its  application  is  limited  to  the 
sterilization  of  glassware  principally — such,  for  example 
as  flasks,  plates,  small  dishes,  test-tubes,  pipettes — and 
such  metal  instruments  as  are  not  seriously  injured  by 
the  high  temperature. 

Sterilization  by  moist  heat — steam — offers  conditions 
much  more  favorable.  The  penetrating  power  of  the 


STERILIZA  TION  J?  Y  HE  A  T.  59 

steam  is  not  only  more  complete,  but  the  tempera- 
ture at  which  sterilization  is  ordinarily  accomplished  is, 
as  a  rule,  not  destructive  to  the  objects  under  treat- 
ment. This  is  conspicuously  seen  in  the  work  of  the 
laboratory ;  the  culture-media,  composed  in  the  main 
of  decomposable  organic  materials  that  would  be  ren- 
dered entirely  worthless  if  exposed  to  the  dry  method 
of  sterilization,  sustain  no  injury  whatever  when  intel- 
ligently subjected  to  an  equally  effective  sterilization 
with  steam.  The  same  may  be  said  of  cotton  and 
woollen  fabrics,  bedding,  clothing,  etc. 

Aside  from  the  relations  of  the  two  methods  to  the 
materials  to  be  sterilized,  their  action  toward  the  or- 
ganisms to  be  destroyed  is  quite  different.  The  pene- 
trating power  of  steam  renders  it  by  far  the  more  effi- 
cient agent  of  the  two.  The  spores  of  several  organisms 
which  are  killed  by  an  exposure  of  but  a  few  moments 
to  the  action  of  steam,  resist  the  destructive  action  of 
dry  heat  at  a  higher  temperature  for  a  much  greater 
length  of  time. 

These  differences  will  be  strikingly  brought  out  in 
the  experimental  work  on  this  subject.  For  our  pur- 
poses it  is  necessary  to  remember  that  the  two  methods 
have  the  following  applications  : 

The  dry  method,  at  a  temperature  of  150°-180°  C. 
for  one  hour,  is  employed  for  the  sterilization  of  glass- 
ware, such  as  flasks,  test-tubes,  culture-dishes,  pipettes, 
plates,  etc. 

Sterilization  by  steam  is  practised  with  all  culture- 
media,  whether  fluid  or  solid.  Bouillon,  milk,  gelatin, 
agar-agar,  potato,  etc.,  are  under  no  circumstances  to 
be  subjected  to  dry  heat. 

The  manner  in  which  heat  is  employed  in  processes 


60  BACTERIOLOGY. 

of  sterilization  varies  with  circumstances.  When  used 
in  the  dry  form  its  application  is  always  continuous — 
L  e.,  the  objects  to  be  sterilized  are  simply  exposed  to 
the  proper  temperature  for  the  length  of  time  necessary 
to  destroy  all  living  organisms  which  may  be  upon 
them.  With  the  use  of  steam,  on  the  other  hand,  the 
articles  to  be  sterilized  are  frequently  of  such  a  nature 
that  a  prolonged  application  of  heat  might  materially  in- 
jure them.  For  this  and  other  reasons  steam  is  usually 
applied  intermittently  and  for  short  periods  of  time.  The 
principles  involved,  in  the  intermittent  method  of  sterili- 
zation depend  upon  differences  of  resistance  to  heat  which 
the  organisms  to  be  destroyed  are  known  to  possess  at 
different  stages  of  their  development.  During  the  life- 
cycle  of  many  of  the  bacilli  there  is  a  stage  in  which 
the  resistance  of  the  organism  to  the  action  of  both 
chemical  and  thermal  agents  is  much  greater  than  at 
other  stages  of  their  development.  This  increased 
power  of  resistance  appears  when  these  organisms  are 
in  the  spore-  or  resting-stage,  to  which  reference  has 
already  been  made.  When  in  the  vegetative  or  grow- 
ing stage  most  bacteria  are  killed  in  a  short  time  by  a 
relatively  low  temperature ;  whereas,  under  conditions 
which  favor  the  production  of  spores,  the  spores  are 
seen  to  be  capable  of  resisting  very  much  higher  tem- 
peratures for  an  appreciably  longer  time ;  indeed,  spores 
of  certain  bacilli  have  been  encountered  that  retain  the 
power  of  germinating  after  an  exposure  of  from  five  to 
six  hours  to  the  temperature  of  boiling  water.  Such 
powers  of  resistance  have  never  been  observed  in  the 
vegetative  stage  of  development.  These  differences  in 
resistance  to  heat  which  the  spore-forming  organisms 
possess  at  their  different  stages  of  development  are 


STERILIZATION  BY  HEAT.  61 

taken  advantage  of  in  the  process  of  sterilization  by 
steam  known  as  the  fractional  or  intermittent  method, 
and  are  the  essential  feature  of  the  principles  on  which 
the  method  is  based. 

As  culture-media  are  dependent  for  their  usefulness 
upon  the  presence  of  more  or  less  unstable  organic 
compounds,  the  object  aimed  at  in  this  method  is  to 
destroy  the  organisms  in  the  shortest  time  and  with 
the  least  amount  of  heat.  It  is  accomplished  by  sub- 
jecting them  to  the  elevated  temperature  at  a  time 
when  the  bacteria  are  in  the  vegetating  or  growing  stage 
— i.  e.j  the  stage  at  which  they  are  most  susceptible  to 
detrimental  influences.  In  order  to  accomplish  this  it  is 
necessary  that  there  should  exist  conditions  of  tempera- 
ture, nutrition,  and  moisture  which  favor  the  vegetation 
of  the  bacilli  and  the  germination  of  any  spores  that 
may  be  present.  When,  as  in  freshly  prepared  nutrient 
media,  these  surroundings  are  found,  the  spore-forming 
organisms  are  not  only  less  likely  to  enter  the  spore- 
stage  than  when  their  environments  are  less  favorable 
to  their  vegetation,  but  spores  which  may  already  exist 
develop  very  quickly  into  mature  cells. 

It  is  plain,  then,  that  with  the  first  application  of 
steam  to  the  substance  to  be  sterilized  the  mature  vege- 
tative forms  are  destroyed;  while  certain  spores  that 
may  be  present  resist  this  treatment,  providing  the 
sterilization  is  not  continued  for  too  long  a  time.  If 
now  the  sterilization  is  discontinued,  and  the  material 
which  presents  conditions  favorable  to  the  germination 
of  the  spores  is  allowed  to  stand  for  a  time,  usually 
for  about  twenty-four  hours,  at  a  temperature  of  from 
20°  to  30°  C.,  those  spores  which  resisted  the  action  of 
the  steam  will,  in  the  course  of  this  interval,  germinate 


62  BACTERIOLOGY. 

into  the  less  resistant  vegetative  cells.  A  second  short 
exposure  to  the  steam  kills  these  forms  in  turn,  and 
by  a  repetition  of  this  process  all  bacteria  that  were 
present  may  be  destroyed  without  the  application  of 
the  steam  having  been  of  long  duration  at  any  time. 
It  should  be  remembered  that  while  spores  which 
may  be  present  are  not  directly  killed  by  such  an 
exposure  to  heat  as  they  experience  in  the  intermit- 
tent method  of  sterilization,  still  their  power  of  ger- 
mination is  somewhat  inhibited  by  this  treatment.  In 
this  method,  therefore,  if  the  temperature  of  100°  C. 
be  employed  for  too  long  a  time,  it  is  possible  so  to 
retard  the  germination  of  the  spores  as  to  render  it 
impossible  for  them  to  develop  into  the  vegetative  stage 
during  the  interval  between  the  heatings.  By  exces- 
sively long  exposures  to  high  temperature,  but  not  long 
enough  to  destroy  the  spores  directly,  the  object  aimed 
at  in  the  method  may  be  defeated, 'and  in  the  end  the 
substance  undergoing  sterilization  be  found  still  to  con- 
tain living  bacteria.  In  this  process  the  plan  that  has 
given  most  satisfactory  results  is  to  subject  the  materials 
to  be  sterilized  to  the  action  of  steam,  under  the  ordi- 
nary conditions  of  atmospheric  pressure,  for  fifteen  min- 
utes on  each  of  three  successive  days,  and  during  the 
intervals  to  maintain  them  at  a  temperature  of  about 
25°-30°  C.  At  the  end  of  this  time  all  living  organ- 
isms which  were  present  will,  as  a  general  rule,  have 
been  destroyed,  and,  unless  opportunity  is  given  for 
the  access  of  new  organisms  from  without,  the  sub- 
stances thus  treated  remain  sterile.  As  an  exception 
to  this,  certain  species  of  spore-forming  bacteria  are 
occasionally  encountered  that  are  not  readily  destroyed 
by  this  mode  of  treatment.  They  are,  presumably, 


STERILIZATION  BY  HEAT.  63 

of  the  group  of  so-called  soil  o/v/r//>/x///.s',  and  repre- 
sent the  forms  most  resistant  to  the  influence  of 
heat.  We  are  not  as  yet  sufficiently  familiar  with 
all  their  peculiarities  to  warrant  our  speaking  with 
certainty  as  to  a  means  of  sterilizing  media  in  which 
they  are  present.  It  does  not  seem  unlikely  that  they  are 
of  the  thermophilic  (possibly  facultative  thermophilic) 
variety,  and  therefore  show  little  tendency  to  develop 
into  the  vegetative  stage  between  the  heatings,  germinat- 
ing perhaps  so  slowly  at  the  temperature  under  which 
they  find  themselves  as  not  to  leave  completely  the  spore- 
stage  before  another  exposure  to  the  steam,  but  mani- 
festing after  a  time  properties  of  life  in  media  that  are 
thought  to  be  sterile  and  which  have  been  placed  aside 
for  subsequent  use.  This  is  a  mere  hypothesis,  however, 
and  is  as  yet  entirely  wanting  in  experimental  proof. 

Theobald  Smith  *  has  recently  called  attention  to  an  in- 
structive personal  experience.  He  finds  that  when  media 
are  present  in  vessels  in  only  thin  layers  the  spores  of; 
"Kdi'robic  species  do  not  develop  into  the  vegetative 
forms  during  the  interval  between  the  heatings,  for  the* 
reason  that  the  shallow  layer  of  medium  does  not  suf- 
ficiently exclude  free  oxygen  to  permit  it ;  and  by  sub- 
jecting such  materials,  apparently  sterilized  by  the  inter- 
mittent method,  to  strictly  anaerobic  conditions  a  devel- 
opment of  anaerobic  species  will  often  occur.  On  the 
other  hand,  if  the  vessels  be  nearly  filled  with  media, 
and  especially  if  the  area  of  the  surface  be  small,  the 
conditions  are  much  more  favorable  to  the  germination 
of  anaerobic  spores  and  sterilization  by  this  process  is 
usually  perfect. 

1  Theobald  Smith  :    Journal  of  Experimental  Medicine,  vol.  iii.  No. 
6,  p.  647. 

UKV  '  J 


64  S  A  CTERIOL  0  G  Y. 

Fortunately,  these  undesirable  experiences  are  rare, 
but  that  they  do  occur,  and  result  in  no  small  degree 
of  annoyance,  will  be  admitted  by  most  bacteriologists. 

It  must  be  borne  in  mind  that  this  method  of  steril- 
ization is  only  applicable  in  those  cases  which  present 
conditions  favorable  to  the  germination  of  the  spores 
into  mature  vegetative  cells.  Dry  substances,  such  as 
instruments,  bandages,  apparatus,  etc.,  or  organic  ma- 
terials in  which  decomposition  is  far  advanced,  where 
conditions  of  nutrition  favorable  to  the  germination  of 
spores  are  not  present,  do  not  offer  the  conditions  requi- 
site for  the  successful  operation  of  the  principles  under- 
lying the  intermittent  method  of  sterilization. 

The  process  of  fractional  sterilization  at  low  tempera- 
tures is  based  upon  exactly  the  same  principle,  but 
differs  in  two  respects  from  the  foregoing  in  the 
manner  by  which  it  is  practised,  viz.,  it  requires  a 
greater  number  of  exposures  for  its  accomplishment, 
and  the  temperature  at  which  it  is  conducted  is  not 
above  68°-70°  C.  It  is  employed  for  the  sterilization  of 
easily  decomposable  materials,  which  would  be  rendered 
useless  by  steam,  but  which  are  unaltered  by  the  tem- 
perature employed,  and  for  certain  albuminous  culture- 
media  that  it  is  desirable  to  retain  in  a  fluid  condition 
during  sterilization,  but  which  would  be  coagulated  if 
exposed  to  higher  temperatures.  This  process  requires 
that  the  material  to  be  sterilized  should  be  subjected  to 
a  temperature  of  68°-70°  C.  for  one  hour  on  each  of 
six  successive  days,  the  interval  of  twenty -four  hours 
between  the  exposures  admitting  of  the  germination 
of  spores  into  mature  cells.  During  this  interval  the 
substances  under  treatment  are  kept  at  about  25°— 


STERILIZATION  BY  HEAT.  65 

30°  C.  The  temperature  employed  in  this  process 
suffices  to  destroy,  in  about  one  hour,  the  vitality  of 
almost  all  organisms  in  the  vegetative  stage.  Formerly 
blood-serum  was  always  sterilized  by  the  intermittent 
method  at  a  low  temperature. 

Sterilization  by  steam  is  also  practised  by  what  may 
be  called  the  direct  method — that  is  to  say,  both  the 
mature  organisms  and  the  spores  which  may  be  present 
in  the  material  to  be  sterilized  are  destroyed  by  a  single 
exposure  to  the  steam.  In  this  method  steam  at  its  ordi- 
nary temperature  and  pressure — live  steam  or  streaming 
steam,  as  it  is  called — is  employed  just  as  in  the  first 
method  described ;  but  it  is  allowed  to  act  for  a  much 
longer  time,  usually  for  not  less  than  an  hour ;  or  steam 
under  pressure,  and  consequently  of  a  higher  tempera- 
ture, is  now  frequently  employed.  By  the  latter  pro- 
cedure a  single  exposure  of  fifteen  minutes  is  sufficient 
for  the  destruction  of  practically  all  bacilli  and  their 
spores,  providing  the  pressure  of  the  steam  is  not  less 
than  one  atmosphere  over  and  above  that  of  normal; 
this  is  approximately  equivalent  to  a  temperature  of 
122°  C.  to  which  the  organisms  are  exposed. 

The  objection  that  has  been  urged  to  both  of  these 
methods,  particularly  that  in  which  steam  under  press- 
ure is  employed,  is  that  the  properties  of  the  media 
are  altered.  Gelatin  is  said  to  become  cloudy  and  lose 
the  property  of  solidifying ;  in  bouillon  and  agar-agar 
fine  precipitates  are  said  to  result,  and  some  believe 
the  reaction  undergoes  a  change.  In  the  experience 
of  those  who  have  used  steam  under  pressure  not  ex- 
ceeding one  or  one  and  one-half  atmospheres  for  ten 
to  fifteen  minutes  these  obstacles  have  rarely  been 


66  BACTERIOLOGY. 

encountered.  There  is  one  point  to  be  borne  in  mind, 
however,  in  using  steam  under  pressure,  viz.,  it  is  not 
possible  to  regulate  the  time  of  exposure  to  the  same 
degree  of  nicety  as  where  ordinary  live  steam  is  used. 
The  reason  for  this  is  that  if  the  apparatus  be  opened 
to  remove  the  objects  being  sterilized  while  the  steam 
within  it  is  under  pressure,  the  escape  of  steam  will 
be  so  rapid  that  all  fluids  within  the  chamber,  thus 
suddenly  relieved  of  pressure,  will  begin  to  boil  vio- 
lently, and,  as  a  rule,  will  boil  quite  out  of  the  tubes, 
flasks,  etc.,  containing  them.  For  this  reason  the 
apparatus  must  be  kept  closed  until  cool,  or  until  the 
gauge  indicates  that  pressure  no  longer  exists  within 
the  chamber,  and  even  then  it  should  be  opened  very 
cautiously.  It  is  patent  from  this  that  the  tempera- 
ture and  time  of  exposure  of  articles  sterilized  by  this 
process  cannot  usually  be  controlled  with  accuracy. 
It  requires  some  time  to  reach  a  given  pressure  after 
the  apparatus  is  closed,  and  it  also  requires  time  for 
cooling  after  the  desired  exposure  to  such  pressure 
before  the  apparatus  can  be  opened. 

It  is  manifest  that  during  these  three  periods,  viz., 
(a)  reaching  the  pressure  desired,  (b)  time  during  which 
the  pressure  is  maintained,  and  (c)  time  for  fall  of  press- 
ure before  the  chamber  can  be  opened,  it  is  difficult  to 
say  certainly  to  what  temperature  and  pressure  the  arti- 
cles in  the  apparatus  have,  on  the  whole,  been  subjected. 
Clearly,  if  the  desired  pressure  and  temperature  have 
been  maintained  for  ten  minutes,  one  cannot  say  that 
this  is  all  the  heat  to  which  the  articles  have  been  sub- 
jected during  their  stay  in  the  chamber.  In  this  light, 
while  steam  under  pressure  may  answer  very  well  for 
routine  sterilization,  still  it  presents  insurmountable 


STERILIZATION -BY  HEAT. 


67 


obstacles  to  its  use  in  more  delicate  experiments  where 
time-exposure  to  definite  temperature  is  of  importance. 
Nevertheless,  for  general  laboratory  purposes,  sterili- 
zation by  steam  under  pressure  has  so  much  to  recom- 
mend it  in  the  way  of  economy  of  time  and  certainty 
of  accomplishment  that  it  has  practically  superseded 
the  older  methods  of  sterilization  by  steaming  or  live 


FIG.  s. 


Steam  sterilizer,  pattern  of  Koch. 

.-team  ;  and  in  most  laboratories  the  original  styles  of 
>  tea  in  sterilizers  are  rapidly  giving  way  to  some  one  or 
another  of  the  modern  forms  of  autoclave. 


For  sterilization  by  live  steam  the  apparatus  com- 
monly employed  has,  until  recently,  been  the  cylin- 
drical boiler  recommended  by  Koch.  (See  Fig.  8.) 


68  BACTERIOLOGY. 

Its  construction  is  very  simple,  essentially  that  of  the 
ordinary  domestic  potato-steamer.  It  consists  of  a 
copper  cylinder,  the  lower  fifth  of  which  is  some- 
what larger  in  circumference  than  the  remaining  four- 
fifths  and  acts  as  a  reservoir  for  the  water  from  which 
the  steam  is  to  be  generated.  Covering  this  section  of 
the  cylinder  is  a  wire  rack  or  grating  through  which 
the  steam  passes,  and  which  serves  to  support  the 
articles  to  be  sterilized.  Above  this,  comprising  the 
remaining  four-fifths  of  the  cylinder,  is  the  chamber 
for  the  reception  of  the  materials  over  and  through 
which  the  steam  is  to  pass.  The  cylinder  is  closed  by 
a  snugly  fitting  cover  through  which  are  usually  two 
perforations  into  which  a  thermometer  and  a  manometer 
may  be  inserted.  The  whole  of  the  outer  surface  of 
the  apparatus  is  encased  in  a  non-conducting  mantle  of 
asbestos  or  felt. 

The  water  is  heated  by  a  gas-flame  placed  in  an  en- 
closed chamber,  upon  which  the  apparatus  rests,  which 
serves  to  diminish  the  loss  of  heat  and  deflection  of  the 
flame  through  the  action  of  draughts.  The  apparatus 
is  simple  in  construction,  and  the  only  point  which  is 
to  be  observed  while  using  it  is  the  level  of  the  water 
in  the  reservoir.  Oji  the  reservoir  is  a  water-gauge 
which  indicates  at  all  times  the  amount  of  water  in  the 
apparatus.  The  amount  of  water  should  never  be  too 
small  to  be  indicated  by  the  gauge  ;  otherwise  there  is 
danger  of  the  reservoir  becoming  dry  and  the  bottom 
of  the  apparatus  being  destroyed  by  the  direct  action  of 
the  flame. 

A  sterilizer  that  has  come  into  very  general  use  in 
bacteriological  laboratories  is  one  originally  intended 
for  use  in  the  kitchen.  It  is  called  the  "  Arnold  steam 


STERILIZATION  UNDER  PRESSURE.  69 

sterilizer."     It  is  very  ingenious  in  its  construction  as 
wi'll  as  economical  in  its  employment. 

The  difference  between  this  apparatus  and  that  just 
described  is  that  it  provides  for  the  condensation  of  the 
steam  after  its  escape  from  the  sterilizing  chamber,  and 
returns  the  water  of  condensation  automatically  to  the 

FIG.  9. 


Arnold  steam  sterilizer. 

reservoir,  so  that  in  practice  the  apparatus  requires  but 
little  attention,  as  with  ordinary  care  there  is  no  likeli- 
hood of  the  water  in  the  reservoir  becoming  exhausted, 
with  the  consequent  destruction  of  the  sterilizer. 
Fig.  9  shows  a  section  through  this  apparatus. 

STERILIZATION   UNDER    PRESSURE. 

The  advantages  of  the  use  of  steam  under  pressure 
for  the  purposes  of  sterilization  have  received  such 
general  recognition  that  almost  everywhere  this  method 


70 


BACTERIOLOGY. 


is  supplanting  the  older  one  of  intermittent  sterilization 
with  steaming  or  live  steam.  By  this  plan  one  is  able 
to  accomplish,  by  a  single  exposure  of  fifteen  minutes  to 
steam  under  a  pressure  of  one  atmosphere,  the  same  end 
that  would,  with  steaming  steam,  require  three  exposures 
of  fifteen  minutes  on  each  of  three  successive  days. 

FIG.  10. 


Autoclave,  pattern  of  Wiesnegg.    A.  External  appearance.    B.  Section. 

For  sterilization  by  steam  under  pressure  several  spe- 
cial forms  of  apparatus  exist.  The  principles  involved 
in  them  all  are,  however,  the  same.  They  provide  for 
the  generation  of  steam  in  a  chamber  from  which  it 
cannot  escape  when  the  apparatus  is  closed.  Upon  the 
cover  of  this  chamber  is  a  safety-valve,  which  can  be 
regulated  so  that  any  degree  of  pressure  (and  coinci- 
dently  of  temperature)  that  is  desirable  may  be  main- 


STERILIZATION  BY  HOT  AIR.  71 

tained  within  the  sterilizing  chamber.    These  sterilizers 
are  known  as  "  digesters  "  and  as  "  autoclaves."     Their 


FIG.  11. 


Autoclave  or  digester  for  sterilizing  by  steam  under  pressure. 

construction   can  best  be  understood   by  reference  to 
Figs.  10  and  11. 

STERILIZATION    BY    HOT   AIR. 

The  hot-air  sterilizers  used  in  laboratories  are  sim- 
ply double-walled  boxes  of  Russian  or  Swedish   iron 


72 


BACTERIOLOGY. 


(Fig.  12),  having  a  double- walled  door,  which  closes 
tightly,  and  a  heavy  copper  bottom.  They  are  pro- 
vided with  openings  for  the  escape  of  the  contained  air 
and  the  entrance  of  the  heated  air.  The  flame,  usually 
from  a  rose-burner  (Fig.  13),  is  applied  directly  to  the 
bottom.  The  heat  circulates  from  the  lower  surface 
around  about  the  apparatus  through  the  space  between 
its  walls. 

FIG.  12. 


FIG.  13. 


Laboratory  hot-air  sterilizer. 


Rose-burner. 


The  construction  of  the  copper  bottom  of  the  appa- 
ratus upon  which  the  flame  impinges  is  designed  to  pre- 
vent the  direct  action  of  the  flame  upon  the  sheet-iron 
bottom  of  the  chamber.  It  consists  of  several  copper 
plates  placed  one  above  the  other,  but  with  a  space  of 
about  4  to  5  mm.  between  the  plates.  These  copper 
bottoms  after  a  time  become  burned  out,  and  unless 


CHEMICAL  STERILIZATION  AND  DISINFECTION.   73 

they  are  replaced  the  apparatus  is  useless.  The  older 
forms  of  hot-air  sterilizers  are  so  constructed  that  their 
repair  is  a  matter  involving  some  time  and  expense. 
To  meet  this  objection  I  have  had  constructed  a  steril- 
izer in  all  respects  similar  to  the  old  form  except  in 
the  arrangement  of  the  copper  bottom.  This  is  made 
in  such  a  way  that  it  can  be  easily  removed,  so  that 
by  keeping  several  sets  of  copper  plates  on  hand  a 
new  plate  can  readily  be  inserted  when  the  old  one  is 
burned  out. 

In  the  employment  of  the  hot-air  sterilizer  care 
should  always  be  given  to  the  condition  of  the  copper 
bntrom  ;  for  the  direct  application  of  heat  to  the  sheet- 
iron  plate  upon  which  the  substances  to  be  steril- 
ized stand  results  not  only  in  destruction  of  the  appa- 
ratus, but  frequently  in  destruction  of  the  substances 
undergoing  sterilization. 

Since  the  temperature  at  which  this  form  of  steril- 
ization is  usually  accomplished  is  high,  from  150°  to 
180°  C.,  it  is  well  to  have  the  apparatus  encased  in 
asbestos  boards,  to  diminish  the  radiation  of  heat  from 
its  surfaces.  This  not  only  confines  the  heat  to  the 
apparatus,  but  guards  against  the  destructive  action  of 
the  radiated  heat  on  woodwork,  furniture,  etc.,  that 
may  be  in  the  neighborhood. 

CHEMICAL,   STERILIZATION   AND   DISINFECTION. 

As  has  been  stated,  it  is  possible  by  means  of  cer- 
tain chemical  substances  to  destroy  all  bacteria  and 
their  spores  that  may  be  within  or  upon  various  mate- 
rials and  objects — i.  e.,  to  sterilize  them  ;  and  it  is  also 
possible  by  the  same  means  to  rob  objects  of  their 


74  BACTERIOLOGY. 

dangerous  infective  properties  without  at  the  same 
time  sterilizing  them — i.  e.,  to  disinfect  them.  This 
latter  process  depends  upon  the  fact  that  the  vital- 
ity of  many  of  the  less  resistant  pathogenic  organ- 
isms is  easily  destroyed  by  an  exposure  to  particular 
chemical  substances  that  may  be  without  effect  upon 
the  more  resistant  saprophytes  and  their  spores  that  are 
present. 

In  general,  the  use  of  chemicals  for  sterilization  is 
not  to  be  considered  in  connection  with  substances  that 
are  to  be  employed  as  culture-media,  and  their  employ- 
ment is  restricted  in  the  laboratory  to  materials  that 
are  of  no  further  value,  and  to  infected  articles  that  are 
not  injured  by  the  action  of  the  agents  used,  though  ex- 
ceptionally such  volatile  germicides  as  chloroform  and 
ether  are  employed  for  the  sterilization  of  special  culture- 
media.  (See  Preservation  of  Blood-serum  with  Chloro- 
form.) In  short,  they  are  mainly  of  value  in  rendering 
infected  waste -materials  innocuous.  For  the  successful 
performance  of  this  form  of  disinfection  there  is  one 
fundamental  rule  always  to  be  borne  in  mind,  viz.,  it 
is  absolutely  essential  to  success  that  the  disinfectant 
used  should  come  in  direct  contact  with  the  bacteria  to 
be  destroyed,  otherwise  there  is  no  disinfection. 

For  this  reason,  one  should  always  remember,  in 
selecting  the  disinfecting  agent,  the  nature  of  the  mate- 
rials containing  the  bacteria  upon  which  it  is  to  act,  for 
the  majority  of  disinfectants,  and  particularly  those  of 
an  inorganic  nature,  vary  in  the  degree  of  their  potency 
with  the  chemical  nature  of  the  mass  to  which  they  are 
applied.  Often  the  materials  containing  the  bacteria 
to  be  destroyed  are  of  such  a  character  that  they  com- 
bine with  the  disinfecting  agent  to  form  insoluble  pre- 


CHEMICAL  STERILIZATION  A$D  DISINFECTION.    75 

cipitates ;  these  so  interfere  with  the  penetration  of  the 
disinfectant  that  many  bacteria  may  escape  its  destruc- 
tive action  entirely  and  no  disinfection  be  accomplished, 
though  an  agent  may  have  been  employed  that  would, 
under  other  circumstances,  have  given  entirely  satisfac- 
tory results. 

In  the  destruction  of  bacteria  by  means  of  chemical 
substances  there  occurs,  most  probably,  a  definite  chem- 
ical reaction — that  is  to  say,  the  characteristics  of  both 
the  bacteria  and  the  agent  employed  in  their  destruction 
are  lost  in  the  production  of  an  inert  third  body,  the 
result  of  their  combination.  It  is  impossible  to  say 
with  absolute  certainty,  as  yet,  that  this  is  the  case ;  but 
the  evidence  that  is  rapidly  accruing  from  the  more 
recent  studies  upon  disinfectants  and  their  mode  of 
action  points  strongly  to  the  accuracy  of  this  belief. 
This  reaction,  in  which  the  typical  structures  of  both 
bodies  concerned  are  lost,  takes  place  between  the  agent 
employed  for  disinfection  and  the  protoplasm  of  the 
bacteria.  For  example,  in  the  reaction  that  is  seen  to 
take  place  between  the  salts  of  mercury  and  albumi- 
nous bodies  there  results  a  third  compound,  which  has 
neither  all  the  characteristics  of  mercury  nor  of  albumin, 
but  partakes  of  the  peculiarities  of  both  ;  it  is  a  combi- 
nation of  albumin  and  mercury,  commonly  known  by  the 
indefinite  term  "  albuminate  of  mercury."  Some  such 
reaction  as  this  apparently  occurs  when  the  soluble  salts 
of  mercury  are  brought  in  contact  with  bacteria.  This 
view  has  been  strengthened  by  the  experiments  of 
Geppert,  in  which  the  reaction  was  caused  to  take  place 
In-tween  the  spores  of  the  anthrax  bacillus  and  a  solu- 
tion of  mercuric  chloride,  the  result  being  the  apparent 
destruction  of  the  vitality  of  the  spores  by  the  forma- 


76  BACTERIOLOGY. 

tion  of  this  third  compound.  In  these  experiments  it 
was  shown  that  though  this  combination  had  taken 
place,  still  it  did  not  of  necessity  imply  the  death  of 
the  spores,  for  if  by  proper  means  the  combination  of 
mercury  with  their  protoplasm  was  broken  up,  many 
of  the  spores  resumed  their  vitality,  with  all  their  pre- 
vious disease-producing  and  cultural  peculiarities.  Gep- 
pert  employed  a  solution  of  ammonium  sulphide  for 
the  purpose  of  destroying  the  combination  of  spore- 
protoplasm  and  mercury ;  the  mercury  was  precipi- 
tated from  the  protoplasm  as  an  insoluble  sulphide, 
and  the  protoplasm  of  the  spores  returned  to  its  original 
condition.  These  and  other  somewhat  similar  experi- 
ments have  given  a  new  impulse  to  the  study  of  disin- 
fectants, and  in  the  light  shed  by  them  many  of  our 
previously  formed  ideas  concerning  the  action  of  dis- 
infecting agents  must  be  modified. 

The  process  of  disinfection  is  not  a  catalytic  one — 
i.  e.9  occurring  simply  as  a  result  of  the  presence  of  the 
disinfecting  body,  which  is  not  itself  decomposed  during 
its  process  of  destruction — but  is,  as  said,  a  definite  chem- 
ical reaction  occurring  within  more  or  less  fixed  limits ; 
that  is  to  say,  with  a  given  amount  of  the  disinfect- 
ant employed  so  much  work,  expressed  in  terms  of  disin- 
fection— destruction  of  bacteria — can  be  accomplished. 

Another  point  in  favor  of  this  view  is  the  increased 
energy  of  the  reaction  with  elevation  of  temperature. 
Just  as  in  many  other  chemical  phenomena  the  inten- 
sity and  rapidity  of  the  reaction  become  greater  under 
the  influence  of  heat,  so  in  the  process  of  disinfection 
the  combination  between  the  disinfectant  and  the  organ- 
isms to  be  destroyed  is  much  more  energetic  at  a  tem- 
perature of  37°  to  39°  C.  than  it  is  at  12°  to  15°  C, 


CHEMICAL  STERILIZATION  AN^D  DISINFECTION.   77 

A  number  of  important  and  novel  suggestions  with 
regard  to  the  modm  operandi  of  disinfection  have  re- 
cently been  brought  out  through  the  work  of  Kronig 
and  Paul,1  who  took  up  the  subject  from  its  physical- 
chemical  standpoint.  The  comprehensive  nature  of  this 
elaborate  investigation  precludes  more  than  a  brief  men- 
tion of  some  of  the  conclusions  reached ;  and  in  order 
that  these  may  be  intelligible  certain  beliefs  (working 
hypotheses)  of  the  physical-chemists  should  be  borne  in 
mind.  In  1887  Arrhenius  proposed  the  theory  that 
when  an  electrolyte  (a  compound  decomposable  by  an 
electric  current)  is  dissolved  in  water  its  molecules  break 
down,  not  simply  into  their  component  atoms,  but  into 
ions,  which  are  atoms  or  groups  of  atoms  having  electro- 
positive and  electro-negative  characteristics.  According 
to  this  theory,  salts,  when  dissolved  in  water,  undergo 
electrolytic  dissociation  into  metallic  and  acidic  ions, 
the  former  being  the  electro-positive  cation,  the  latter 
the  electro-negative  anion  ;  sodium  chloride,  for  exam- 
ple, resolving  itself,  under  these  conditions,  into  its 
sodium,  or  metal-ion,  and  its  chlorine,  or  acidic  ion. 
The  electro-positive  cations,  according  to  Ostwald,  com- 
prise the  metals  and  metal-like  radicals,  such  as  am- 
monium (NH4)  and  hydrogen  (H) ;  while,  the  electro- 
negative anions  include  the  halogens,  the  acidic  radicals 
(such  as  NO3  and  SO4),  and  hydroxyl.2  Using  this  theory 
as  the  basis  of  their  investigations,  Kronig  and  Paul 

1  Kronig  and  Paul:    Zeitschrift  fur  Hygiene  und  Infectionskrank- 
heiten,  1897,  Band  xxv.  S.  1-112. 

2  Consult  Ostwald's  Lehrbuch  der  Allg.  Chemic  ;    or  Muir's  transla- 
tion of  Ostwald's  Solutions,  p.  189,  published  by  Longmans,  Green  & 
<'(,..  London  and  New  York,  1891.    Also  "  The  Rise  of  the  Theory  of 
Kltvtrolytic  Dissociation,"  etc.,  by  H.  C.  Jones,  Ph.  D.,  Johns  Hopkins 
Hospital  Bulletin,  No.  87,  June,  1898,  p.  136. 


78  BACTERIOLOGY. 

reached  the  following  conclusions  with  regard  to  the 
action  of  chemical  disinfectants  : 

The  germicidal  value  of  a  metallic  salt  depends  not 
only  upon  its  specific  character,  but  also  upon  that  of  its 
anion.  % 

Solutions  of  metallic  salts  in  which  the  metallic  part 
is  represented  by  a  complex  ion  and  in  which  the  con- 
centration of  the  metal  ion  is  very  slight,  have  but 
feeble  disinfecting  activity. 

The  halogen  compounds  of  mercury  act  according  to 
the  degree  of  their  dissociation. 

The  disinfecting  poAver  of  the  halogens — chlorine, 
bromine,  iodine — (as  well  as  their  compounds)  is  in  in- 
verse ratio  to  their  atomic  weights. 

The  disinfecting  activity  of  watery  solutions  of  mer- 
curic chloride  is  diminished  by  the  addition  to  them 
of  other  halogen  compounds  of  metals  and  of  hydro- 
chloric acid.  It  appears  probable  that  this  is  due  to 
obstruction  offered  to  electrolytic  dissociation. 

The  disinfecting  activities  of  watery  solutions  of  mer- 
curic nitrate,  mercuric  sulphate,  and  mercuric  acetate  are 
increased  by  the  moderate  addition  of  sodium  chloride. 

In  general,  acids  disinfect  according  to  the  degree 
of  their  dissociation — i.  e.,  according  to  the  concentra- 
tion of  their  hydrogen  ions  in  the  solution. 

The  bases,  potassium,  sodium,  lithium,  and  ammo- 
nium hydroxide,  disinfect  according  to  the  degree  of 
their  dissociation — i.  e.,  corresponding  to  the  concen- 
tration of  their  hydroxyl  ions  in  the  solution. 

The  disinfecting  activity  of  metallic  salts  is,  in  gen- 
eral, less  in  albuminous  fluids  than  in  water.  .  It  is 
probable  that  this  is  due  to  a  diminution  in  the  concen- 
tration of  metallic  ions  in  the  solution. 


CHEMICAL  STERILIZATION  AND  DISINFECTION.   79 

What  has  been  said  refers  more  particularly  to  the 
inorganic  salts  which  are  employed  for  this  purpose. 
It  is  probable  that  the  organic  bodies  possessing  dis- 
infectant properties  owe  this  power  to  some  such  similar 
rcuction,  though,  as  yet,  these  substances  have  not  been 
so  thoroughly  studied  in  this  relation. 

The  reaction  between  the  inorganic  salts  and  albu- 
minous bodies  is  not  selective ;  they  combine  in  most 
instances  with  any  or  all  protoplasmic  bodies  present. 
For  this  reason  the  employment  of  many  of  the  com- 
moner disinfectants  in  general  practice  is  a  matter  of 
doubtful  advantage.  For  example,  the  disinfection  of 
excreta,  sputum,  or  blood,  containing  pathogenic  organ- 
isms, by  means  of  corrosive  sublimate,  is  a  procedure 
of  questionable  success.  The  amount  of  sublimate  em- 
ployed may  be  entirely  used  up  and  rendered  inactive 
as  a  disinfectant  by  the  ordinary  protoplasmic  sub- 
stances present,  without  having  any  appreciable  effect 
upon  the  bacteria  which  may  be  in  the  mass. 

These  remarks  are  introduced  in  order  to  guard 
against  the  implicit  confidence  so  often  placed  in  the 
disinfecting  value  of  corrosive  sublimate.  In  many 
bacteriological  laboratories  it  is  the  custom  to  keep  at 
hand  vessels  containing  solutions  of  corrosive  sublimate, 
into  which  infectious  materials  may  be  placed.  The 
value  of  this  procedure,  as  we  have  just  learned,  may 
be  more  or  less  questionable,  especially  in  those  cases 
in  which  the  substance  to  be  disinfected  is  of  a  proteid 
nature  and  where  the  solution  used  is  not  freshly  pre- 
pared. On  the  introduction  of  such  substances  into 
the  sublimate1  solution  the  mercury  is  quickly  precip- 
itated by  the  albumin,  and  its  disinfecting  properties 
may  be  in  large  part  or  entirely  destroyed ;  we  may  in 


80  BACTERIOLOGY. 

a  very  short  time  have  little  else  than  water  containing 
a  precipitate  of  albumin  and  mercury,  in  so  far  as  its 
value  as  a  disinfectant  is  concerned. 

Though  the  other  inorganic  salts  have  not  been  so 
thoroughly  studied  in  this  connection,  it  is  nevertheless 
probable  that  the  same  precautions  should  be  taken  in 
their  employment  as  we  now  know  to  be  necessary  in 
the  use  of  the  salts  of  mercury. 

Where  it  is  desirable  to  use  chemical  disinfectants 
in  the  laboratory  much  more  satisfactory  results  can 
usually  be  obtained  from  the  employment  of  carbolic 
acid  in  solution.  A  3  or  4  per  cent,  solution  of  com- 
mercial carbolic  acid  in  water  requires  longer  for  dis- 
infection ;  but  it  is,  at  the  same  time,  open  to  fewer 
objections  than  are  solutions  of  the  inorganic  salts; 
though  here,  too,  we  find  a  somewhat  analogous  reac- 
tion between  the  carbolic  acid  and  proteid  matters. 
Under  ordinary  circumstances  its  action  is  complete 
in  from  twenty  minutes  to  one-half  hour.  It  is  not 
reliable  for  the  disinfection  of  resistant  spores;  such, 
for  instance,  as  those  of  bacillus  anthrads. 

All  tissues  containing  infectious  organisms  should  be 
burned,  and  all  cloths,  test-tubes,  flasks,  and  dishes 
should  be  boiled  in  2  per  cent,  soda  (ordinary  washing- 
soda)  solution  for  fifteen  to  twenty  minutes,  or  placed 
in  the  steam  sterilizer  for  half  an  hour. 

Intestinal  evacuations  may  best  be  disinfected  with 
boiling  water  or  with  milk  of  lime,  a  mixture  composed 
of  lime  in  solution  and  in  suspension — ordinary  fluid 
"  white-wash."  This  should  be  thoroughly  mixed  with 
the  evacuations  until  the  mass  reacts  distinctly  alkaline, 
and  should  remain  in  contact  with  them  for  one  or 
two  hours.  Excreta  may  also  be  easily  disinfected  by 


CHEMICAL  STERILIZATION  AND  DISINFECTION.   81 

thoroughly  mixing  them  with  two  or  three  times  their 
volume  of  boiling  water,  after  which  they  are  kept  cov- 
ered until  cool. 

Sputum  in  which  tubercle  bacilli  are  present,  as  well 
as  the  vessel  containing  it,  must  be  boiled  in  2  per  cent, 
soda  solution  for  fifteen  minutes,  or  steamed  in  the  ster- 
ilizer for  at  least  half  an  hour. 

On  the  whole,  in  the  laboratory  we  should  as  yet 
rely  more  upon  the  destructive  properties  of  heat  than 
upon  those  of  chemical  agents. 

From  what  has  been  said,  the  absurdity  of  sprink- 
ling here  and  there  a  little  carbolic  acid,  or  in  placing 
vessels  of  carbolic  acid  about  apartments  in  which  in- 
fectious diseases  are  in  progress,  must  be  plain.  Treat- 
ment of  water-closets  and  cesspools  by  allowing  now 
and  then  a  few  cubic  centimetres  of  some  so-called 
disinfectant  to  trickle  through  the  pipes  is  ridiculous. 
A  disinfectant  must  be  applied  to  the  bacteria,  and  must 
be  in  contact  with  them  for  a  long  enough  time  to  insure 
the  destruction  of  their  life. 

In  the  light  of  the  latest  experiments  upon  disin- 
fectants, the  place  formerly  occupied  by  many  agents 
in  the  list  of  substances  employed  for  the  purpose  will 
most  likely  be  changed  as  they  are  studied  more  closely. 
The  agents,  then,  which  will  prove  of  greatest  value  in 
the  laboratory  for  the  purpose  of  rendering  infectious 
materials  harmless  are :  heat,  either  by  burning,  by 
steaming  for  from  half  an  hour  to  an  hour,  or  by  boil- 
ing in  a  2  per  cent,  sodium  carbonate  solution  for  fifteen 
minutes ;  3  to  4  per  cent,  solution  of  commercial  car- 
bolic acid ;  milk  of  lime,  and  a  solution  of  chlorinated 
fimr  ("chloride  of  lime")  containing  not  less  than  0.25 
per  cent,  of  free  chlorine.  The  chloride  of  lime  from 

6 


82  BACTERIOLOGY. 

which  such  a  solution  is  to  be  made  should  be  fresh 
and  of  good  quality.  Good  chlorinated  lime,  as  pur- 
chased in  the  shops,  should  contain  not  less  than  25  to 
30  per  cent,  of  available  chlorine.  The  materials  to  be 
disinfected  in  either  of  the  lime  solutions  should  remain 
in  them  for  about  two  hours.  The  solutions  should  be 
freshly  prepared  when  needed,  as  they  rapidly  decom- 
pose upon  standing. 

An  antiseptic  is  a  body  which,  by  its  presence,  pre- 
vents the  growth  of  bacteria  without  of  necessity  killing 
them.  A  body  may  be  an  antiseptic  without  possessing 
disinfecting  properties  to  any  very  high  degree,  but  a 
disinfectant  is  always  an  antiseptic  as  well. 

A  germicide  is  a  body  possessing  the  property  of 
killing  bacteria. 


CHAPTER   IV. 

Principles  involved  in  the  methods  of  isolation  of  bacteria  in  pure 
culture  by  the  plate  method  of  Koch— Materials  employed. 

As  was  stated  in  the  introductory  chapter,  the  isola- 
tion in  pure  cultures  of  the  different  species  that  may 
be  present  in  mixtures  of  bacteria  was  rendered  possible 
only  through  the  methods  suggested  by  Koch.  Since 
the  adoption  of  these  methods  they  have  undergone 
many  modifications,  but  the  fundamental  principle  re- 
mains the  same.  The  observation  which  led  to  their 
development  was  a  very  simple  one,  and  one  that  is 
frequently  before  us.  Koch  noticed  that  on  solid  sub- 
stances, such,  for  example,  as  a  slice  of  potato  or  of 
bread,  which  had  been  exposed  for  a  time  to  the  air  and 
which  afforded  proper  nourishment  for  the  lower  organ- 
isms, there  developed  after  a  short  time  small  patches 
which  proved  to  be  colonies  of  bacteria.  Each  of 
these  colonies  on  closer  examination  proved  to  be,  as 
a  rule,  composed  of  distinct  species  of  micro-organisms. 
There  was  little  tendency  on  the  part  of  these  colonies 
to  become  confluent,  and  from  the  differences  in  their 
naked-eye  appearances  it  was  easy  to  see  that  they  rep- 
resented, in  the  main,  the  development  of  different 
species  of  bacteria. 

The  question  that  then  presented  itself  was  :  If  from 
a  mixture  of  organisms  floating  in  the  air  it  is  possible 
in  this  way  to  obtain  in  pure  cultures  the  component 
individuals,  what  means  can  be  employed  to  obtain  the 

83 


84  BACTERIOLOGY. 

same  results  at  will  from  mixture  of  different  species  of 
bacteria  when  found  together  under  other  conditions  ? 
It  was  plain  that  the  organisms  were  to  be  distinguished 
primarily,  the  one  from  the  other,  only  by  the  structure 
and  general  appearance  of  the  colonies  growing  from 
them,  for  by  their  morphology  alone  this  is  impossible. 
What  means  might  be  devised,  then,  for  separating  the 
individual  members  of  a  mixture  in  such  a  way  that 
they  would  remain  in  a  fixed  position,  and  be  so  widely 
separated,  the  one  from  the  other,  as  not  to  interfere 
with  the  production  of  colonies  of  characteristic  appear- 
ance, which  would,  under  favorable  conditions,  develop 
from  each  individual  cell  ? 

If  one  take  in  the  hand  a  mixture  of  barley,  rye, 
corn,  oats,  etc.,  and  attempt  to  separate  the  mass  into 
its  constituents  by  picking  out  the  different  grains,  much 
difficulty  is  experienced ;  but  if  the  handful  of  grain  be 
thrown  upon  a  large,  flat  surface,  as  upon  a  table,  the 
grains  become  widely  separated  and  the  task  is  con- 
siderably simplified ;  or,  if  sown  upon  proper  soil,  the 
various  grains  will  develop  into  growths  of  entirely 
different  external  appearance,  by  .which  they  can  readily 
be  recognized  as  unlike  in  nature.  Similarly,  if  a  test- 
tube  of  decomposed  bouillon  be  poured  upon  a  large, 
flat  surface,  the  individual  bacteria  in  the  mass  are 
much  more  widely  separated,  the  one  from  the  other, 
than  they  were  when  the  bouillon  was  in  the  tube  ;  but 
they  are  in  a  fluid  medium,  and  there  is  no  possibility 
of  their  either  remaining  separated  or  of  their  forming 
colonies  under  these  conditions,  so  that  it  is  impossible 
by  this  means  to  pick  out  the  individuals  from  the 
mixture. 

If,  however,  some  substance  could  be  found  which 


METHODS  OF  ISOLATION.  85 

possesses  the  property  of  being  at  one  time  fluid  and 
at  another  time  solid,  and  which  could  be  added  to 
this  bouillon  without  in  any  way  interfering  with  the 
life-functions  of  the  bacteria,  then,  as  solidification  set 
in,  the  organisms  could  be  fixed  in  their  positions,  and 
the  conditions  would  be  analogous  to  those  seen  on  the 
bit  of  potato. 

FIG.  14. 


Showing  certain  macroscopic  characteristics  of  colonies.   Natural  size. 

Gelatin  possesses  this  property,  and  it  was,  therefore, 
used.  At  a  temperature  which  does  not  interfere  with 
the  life  of  the  organisms  it  is  quite  fluid,  whereas  when 
subjected  to  a  lower  temperature  it  solidifies.  When 


86  BACTERIOLOGY. 

once  solid  it  may  be  kept  at  a  temperature  favorable  to 
the  growth  of  the  bacteria  and  will  remain  in  its  solid 
condition. 

Gelatin  was  added  to  the  fluids  containing  mixtures 
of  bacteria,  and  the  whole  was  then  poured  upon  a  large, 
flat  surface,  allowed  to  solidify,  and  the  results  noted. 
It  was  found  that  the  conditions  seen  on  the  slice  of 
potato  could  be  reproduced  ;  that  the  individuals  in  the 
mixture  of  bacteria  grew  well  in  the  gelatin,  and,  as  on 
the  potato,  grew  in  colonies  of  typical  macroscopic  struct- 
ure, so  that  they  could  easily  be  distinguished  the  one 
from  the  other  by  their  naked-eye  appearances.  (See 
Fig.  14.)  It  was  necessary,  however,  to  use  a  more 
dilute  mixture  of  bacteria  than  the  original  decom- 
posed bouillon.  The  number  of  individuals  in  the 
tube  was  so  enormous  that  on  the  gelatin  plate  they 
were  so  closely  packed  together  that  it  was  impossible 
to  pick  them  out,  not  only  because  of  their  proximity 
the  one  to  the  other,  but  also  because  this  packing 
together  materially  interfered  with  the  production  of 
those  characteristic  differences  visible  to  the  naked 
eye.  The  numbers  of  the  organisms  were  then  dimin- 
ished by  a  process  of  dilution,  consisting  of  trans- 
ferring a  small  portion  of  the  original  mixture  into  a 
second  tube  of  sterilized  bouillon  to  which  gelatin  had 
been  added  and  liquefied  ;  from  this  a  portion  was 
added  to  a  third  gelatin-bouillon  tube,  and  so  on. 
These  were  then  poured  upon  large  surfaces  and  allowed 
to  solidify.  The  result  was  entirely  satisfactory.  On 
the  gelatin  plates  from  the  original  tube,  as  was  ex- 
pected, the  colonies  were  too  numerous  to  be  of  use ; 
on  the  plates  made  from  the  first  dilution  they  were 
much  fewer  in  number,  but  usually  they  were  still  too 


METHODS  OF  ISOLATION.  87 

numerous  and  too  closely  packed  to  permit  of  charac- 
teristic growth  ;  on  the  second  dilution  they  were,  as  a 
rule,  fewer  in  number  and  widely  separated,  so  that 
the  individuals  of  each  species  were  in  no  way  pre- 
vented by  the  proximity  of  their  neighbors  from  grow- 
ing each  in  its  typical  way.  (See  Fig.  15.)  There 
was  then  no  difficulty  in  picking  out  the  colonies  result- 
ing from  the  growth  of  the  different  individual  bacteria. 


FIG.  15. 


C 


Series  of  plates  showing  the  results  of  dilution  upon  the  number  of  colonies : 
A.  Plate  No.  1,  or  "  original."  B.  First  dilution,  or  Plate  No.  2.  C.  Second 
dilution,  or  Plate  No.  3.  About  one-fourth  natural  size. 

This,  then,  is  the  principle  underlying  Koch's  method 
for  the  isolation  of  bacteria  by  means  of  solid  media. 

The  fundamental  constituent  of  the  media  employed 
is  the  bouillon,  which  contains  all  the  elements  necessary 
for  the  nutrition  of  most  bacteria,  the  gelatin  being  em- 
ployed simply  for  the  purpose  of  rendering  the  bouillon 
solid.  The  medium  on  which  the  organisms  are  grow- 
ing is,  therefore,  simply  solidified  bouillon,  or  beef- 
tea. 

In  practice,  two  forms  of  gelatin  are  employed — the 
one  an  animal  or  bone  gelatin,  the  ordinary  table  gelatin 
of  good  quality ;  the  other  a  vegetable  gelatin,  known 
as  agar-agar,  the  native  name  for  Ceylon  moss  or  Ben- 


88  BACTERIOLOGY. 

gal  isinglass,  which  is  obtained  from  a  group  of  algae 
growing  in  the  sea  along  the  coast  of  Japan,  China,  and 
many  parts  of  the  East,  where  it  is  employed  as  an 
article  of  diet  by  the  natives. 

Aside  from  these  differences  in  origin  of  the  two 
forms  of  gelatin  employed,  their  behavior  under  the 
influence  of  heat  and  of  bacterial  growth  renders  them 
of  different  application  in  bacteriological  work.  The 
animal  gelatin  liquefies  at  a  much  lower  temperature, 
and  also  requires  a  lower  temperature  for  its  solidifica- 
tion, than  does  the  agar-agar.  Ordinary  gelatin,  in  the 
proportion  commonly  used  in  this  work,  liquefies  at 
about  24°-26°  C.,  and  becomes  solid  at  from  8°  to  10°  C. 
It  may  be  employed  for  those  organisms  which  do  not 
require  a  higher  temperature  for  their  development  than 
22°-24°  C.  Agar-agar,  on  the  other  hand,  does  not 
liquefy  until  the  temperature  has  reached  about  98°— 99° 
C.  It  remains  fluid  ordinarily  until  the  temperature  has 
fallen  to  38°-39°  C.,  when  it  rapidly  solidifies.  For 
our  purposes,  only  that  form  of  agar-agar  can  be  used 
which  remains  fluid  at  from  38°  to  40°  C.  Agar-agar 
which  remains  fluid  only  at  a  temperature  above  this 
point  would  be  too  hot,  when  in  a  fluid  state,  for  use ; 
many  of  the  organisms  introduced  into  it  would  either 
be  destroyed  or  checked  in  their  development  by  so  high 
a  temperature.  Agar-agar  is  employed  in  those  cases  in 
which  the  cultivation  must  be  conducted  at  a  tempera- 
ture above  the  melting-point  of  gelatin. 

In  addition  to  their  differences  when  under  the  in- 
fluence of  various  temperatures,  the  relations  of  these 
two  gelatins  to  bacteria  are  quite  distinct.  Many  bac- 
teria bring  about  alterations  in  gelatin  which  cause  it  to 
become  liquid  (a  process  analogous  to  peptonization),  in 


METHODS  OF  ISOLATION.  89 

which  state  it  remains.    There  are  no  known  organisms 
that  bring  about  such  a  change  in  agar-agar. 

As  a  rule,  the  colony-formations  seen  upon  gelatin 
are  much  more  characteristic  than  those  which  develop 
on  agar-agar,  and  for  this  reason  gelatin  is  to  be  pre- 
ferred when  circumstances  will  permit.  Both  gelatin 
and  agar-agar  may  be  used  in  the  preparation  of  plates 
and  Esmarch  tubes,  subsequently  to  be  described. 


CHAPTER  V. 

Preparation    of  media — Bouillon,   gelatin,   agar-agar,  potato,  blood- 
serum,  etc. 

As  has  been  stated,  the  fundamental  constituent  of 
culture-media  is  beef-tea,  or  bouillon. 

BOUILLON. — The  directions  of  Koch  for  the  prepara- 
tion of  this  medium  have  undergone  many  modifications 
to  meet  special  cases  ;  but  -for  general  use  his  original 
formula  is  still  retained.  It  is  as  follows  :  500  grammes 
of  finely  chopped  lean  beef,  free  from  fat  and  tendons, 
are  to  be  soaked  in  one  litre  of  water  for  twenty-four 
hours,  during  which  time  the  mixture  is  to  remain  in 
an  ice-chest  or  to  be  otherwise  kept  at  a  low  tempera- 
ture. At  the  end  of  twenty-four  hours  it  is  to  be  strained 
through  a  coarse  towel  and  pressed  until  a  litre  of  fluid  is 
obtained.  To  this  are  to  be  added  10  grammes  (1.0  per 
cent.)  of  dried  peptone  and  5  grammes  (0.5  per  cent.)  of 
common  salt  (NaCl).  It  is  then  to  be  rendered  exactly 
neutral  or  very  slightly  alkaline  with  a  few  drops  of 
saturated  sodium  carbonate  solution.  The  flask  con- 
taining the  mixture  is  then  to  be  placed  either  in  a 
steam  sterilizer  or  in  a  water-bath,  or  over  a  free  flame, 
and  kept  at  the  boiling-point  until  all  the  albumin  is 
coagulated  and  the  fluid  portion  is  of  a  clear,  pale 
straw  color.  It  is  then  filtered  through  a  folded  paper 
filter  and  sterilized  in  the  steam  sterilizer  by  the  frac- 
tional method.  Certain  modifications  of  this  method 
are  of  sufficient  value  to  justify  mention.  Most  im- 

90 


BOUILLON.  91 

portant  is  the  neutralization.  Ordinarily,  this  is  ac- 
complished with  the  saturated  sodium  carbonate  solu- 
tion, and  the  reaction  is  determined  with  red  and  blue 
litmus  papers  ;  for  the  beginner  this  method  serves  most 
purposes. 

The  sodium  carbonate  solution  is  not  so  good,  how- 
ever, as  a  strong  solution  of  caustic  soda  or  potash, 
because  the  carbonic  acid  liberated  from  the  sodium 
carbonate  frequently  gives  rise  to  a  confusing,  tem- 
porary acid  reaction  which  disappears  on  heating; 
nor  is  litmus  the  most  reliable  indicator  to  employ. 
To  obviate  this,  Schultz  (Centralb.  /.  Bdkt.  u,  Paramt- 
i-nkt.i.iidcj  1891,  Bd.  x.,  Nos.  2  and  3)  recommends  exact 
titration  with  a  solution  of  caustic  soda.  For  this  pur- 
pose a  4  per  cent,  solution  of  caustic  soda  is  prepared. 
From  this  a  0.4  per  cent,  solution  is  made,  and  witli  it 
the  titration  is  practised.  After  the  bouillon  has  been 
deprived  of  all  coagulable  albumin  and  blood-coloring- 
mutter  by  boiling  and  filtration,  and  has  cooled  down 
to  the  temperature  of  the  air,  its  volume  is  exactly 
measured. 

From  this  a  sample  of  exactly  5  or  10  c.c.  is  then  taken, 
and  to  it  a  few  drops  of  one  of  the  indicators  com- 
monly employed  in  analytical  work  are  added.  Schultz 
recommends  1  drop  of  phenolphtalein  solution  (1 
gramme  of  phenolphtalein  in  300  c.c.  of  alcohol)  to  1 
c.c.  of  bouillon.  The  beaker  containing  the  sample  is 
placed  upon  white  paper,  and  the  dilute  caustic  soda 
solution  is  then  allowed  to  drop  very  slowly  into  it 
from  a  burette,  until  there  appears  a  very  delicate  rose 
color,  which  indicates  the  beginning  of  alkaline  reaction. 
A  second  sample  of  the  bouillon  is  treated  in  the  same 
way.  If  the  amounts  of  caustic  soda  solution  required 


92  BACTERIOLOGY. 

for  each  sample  deviate  but  very  slightly  or  not  at  all 
the  one  from  the  other,  the  mean  of  these  amounts  is 
taken  as  the  amount  of  alkali  necessary  to  neutralize 
the  quantity  of  bouillon  employed.  If  10  c.c.  of  bouillon 
were  employed,  then  for  the  whole  amount  of  1  litre 
just  100  times  as  much,  minus  that  for  the  two  samples 
used  in  titration,  will  be  needed.  For  example :  to 
neutralize  10  c.c.  of  bouillon  2  c.c.  of  the  diluted  (0.4 
per  cent.)  caustic  soda  solution  were  employed.  For 
the  remaining  980  c.c.  of  the  litre  of  bouillon,  then, 
196  c.c.  (200  c.c.  less  4  c.c.,  the  amount  employed  for  the 
two  samples  of  10  c.c.  each  of  bouillon)  are  needed  of 
the  0.4  per  cent,  solution,  or  one-tenth  of  this  amount 
of  the  4  per  cent,  caustic  soda  solution. 

For  the  neutralization  of  the  whole  bulk  of  the 
bouillon  it  is  better  to  employ  the  stronger  alkaline 
solution,  as  by  its  use  the  volume  is  not  increased 
to  so  great  an  extent  as  when  the  dilute  solution  is 
used. 

It  is  evident  that  this  method  is  much  more  exact 
than  that  ordinarily  employed  ;  but  at  the  same  time  it 
must  be  remembered  that  for  its  success  exactness  in 
the  measurement  of  the  volumes  and  in  the  preparation 
of  the  dilutions  is  required.  To  obviate  error,  it  is 
better  to  employ  this  method  when  the  solutions  are  all 
cool  and  of  nearly  the  same  temperature,  so  that  rapid 
fluctuations  in  temperature,  and  consequent  alterations 
in  volume,  will  not  materially  interfere  with  the  accu- 
racy of  the  results. 

This  method  of  neutralization,  as  suggested  by 
.Schultz,  should  be  adopted  for  experiments  in  which  it 
is  desirable  to  have  the  reaction  of  the  medium  accurate 
and  constantly  of  the  same  degree. 


BOUILLO^.  93 

For  the  purposes  of  the  beginner,  however,  results 
quite  satisfactory  in  their  nature  may  be  obtained  by  the 
employment  of  the  saturated  sodium  carbonate  solution 
for  neutralization,  with  litmus  paper  as  the  indicator. 
For  some  time,  however,  it  has  been  our  practice  to 
employ  the  yellow  curcuma  paper  for  the  detection 
of  alkalinity,  rather  than  the  red  litmus  paper. 

In  the  exhaustive  paper  of  Fuller l  on  the  question  of 
reaction  it  was  shown  that  the  results  obtained  by  titrating 
the  same  culture-medium  with  the  same  alkaline  solu- 
tion differed  very  markedly  with  the  indicator  employed. 
For  instance,  a  litre  of  ordinary  meat-infusion  nutrient 
agar-agar  required  47  c.c.  of  a  normal  caustic  alkali 
solution  to  neutralize  it  when  phenolphtalein  was  the 
indicator  used,  28  c.c.  when  blue  litmus  was  employed, 
and  5  c.c.  when  rosolic  acid  was  substituted.  It  is 
manifest  from  this  that  the  actual  reactions  of  media, 
in  the  neutralization  of  which  different  indicators  have 
been  used,  may  differ  very  widely  from  one  another, 
and  that  the  results  of  cultivation  on  a  medium  neu- 
tralized by  one  method  are  not  fairly  comparable  with 
those  obtained  when  another  indicator  has  been  used. 
For  the  sake  of  uniformity  Fuller  suggests  that  bac- 
teriologists should  agree  upon  some  one  trustworthy 
method  of  neutralization  and  employ  it  to  the  exclusion 
of  other  methods*  He  recommends,  as  the  procedure 
that  has  given  the  most  satisfactory  results  in  his  hands, 
a  modification  of  Schultz's  method,  viz.,  5  c.c.  of  the 
culture-medium  are  to  be  mixed  with  45  c.c.  of  distilled 
water  in  a  porcelain  evaporating-dish  and  boiled  for 

1  Fuller :  "  On  the  Proper  Reaction  of  Nutrient  Media  for  Bacterial 
Cultivation,"  Public  .Health  (Journal  of  the  American  Public  Health 
Association),  Quarterly  Series,  1895,  vol.  i.  p.  381. 


94  BACTERIOLOGY. 

three  minutes,  after  which  1  c.c.  of  phenolphtalein 
solution l  is  added  and  titration  with  the  one-twentieth 
normal  caustic  alkali  solution  is  quickly  made.  The 
neutral  point  (slightly  on  the  side  of  alkalinity)  is  indi- 
cated by  the  appearance  of  a  pink  color,  the  effect  of 
the  alkali  on  the  phenolphtalein.  From  the  amount 
of  one-twentieth  normal  alkali  solution  needed  for  5 
c.c.  of  the  medium  it  is  easy  to  calculate  the  number  of 
cubic  centimetres  of  the  normal  solution  that  will  be 
required  to  neutralize  the  entire  mass. 

The  phenolphtalein  neutral  point  lies  so  high,  aver- 
aging 47  c.c  of  normal  caustic  alkali  solution  per  litre 
for  nutrient  meat-infusion  agar-agar,  and  56  c.c.  per 
litre  for  nutrient  gelatin,  that  it  is  improbable  from  ex- 
perience gained  from  the  older  methods  that  the  condi- 
tions offered  by  media  neutral  to  this  indicator  are  suit- 
able for  the  growth  of  all  bacteria,  so  that  with  particular 
species  it  may  be  necessary  to  determine  by  experiment 
the  degree  of  deviation  from  the  neutral  point  that  is 
best  suited  for  development.  In  Fuller's  experience  the 
degree  of  deviation  from  the  phenolphtalein  neutral 
point  that  gives  in  general  the  best  results  is  represented 
by  from  15  to  20  of  his  scale — i.  e.,  there  should  remain 
enough  uncombined  acid  in  a  litre  of  the  finished  me- 
dium to  require  the  further  addition  of  caustic  alkali  to 
the  extent  of  from  15  to  20  c.c.  of  a  normal  solution  to 
bring  the  reaction  of  the  mass  up  to  the  phenolphtalein 
neutral  point..  Thus,  for  example,  if  upon  titration  it 
should  be  found  that  to  neutralize  a  litre  of  nutrient 
meat-infusion  gelatin  by  the  phenolphtalein  process 
55  c.c.  of  normal  caustic  alkali  solution  would  be 
needed,  the  amount  actually  added  would  be  from  35 
1  A  0.5  per  cent,  solution  of  the  powder  in  50  per  cent,  alcohol. 


NUTRIENT  GELATIN. 


to  40  c.c. — i.  e.j  from  15  to  20  c.c.  less  than  the 
amount  needed  to  bring  the  reaction  up  to  the  neutral 
point. 

Not  infrequently  the  filtered  bouillon,  neutralized 
and  sterilized,  will  be  seen  to  contain  a  fine,  flocculent 
precipitate.  This  may  be  due  either  to  excess  of  alka- 
linity or  to  incomplete  precipitation  of  the  albumin. 
The  former  may  be  corrected  with  dilute  acetic  or 
hydrochloric  acid,  and  the  bouillon  again  boiled,  fil- 
tered, and  sterilized  ;  or,  if  due  to  the  latter  cause,  sub- 
sequent boiling  and  filtration  usually  result  in  ridding 
the  bouillon  of  the  precipitate. 

Another  modification  now  generally  employed  is 
the  use  of  meat-extracts  instead  of  infusion  of  meat. 
Almost  any  of  the  meat-extracts  of  commerce  answer 
the  purpose,  though  we  usually  employ  Liebig's.  It 
is  used  in  the  strength  of  from  two  to  four  grammes  to 
the  litre  of  water.  Peptone  and  sodium  chloride  are 
added  as  in  the  bouillon  made  from  meat-infusion. 
The  advantages  of  meat-extract  are  :  it  takes  less  time ; 
affords  a  solution  of  more  uniform  composition  if  used 
in  fixed  proportions ;  and  in  general  use  gives  results 
that  are  equally  as  satisfactory  as  those  obtained  from 
the  employment  of  infusion  of  meat. 

NUTRIENT  GELATIN. — For  the  preparation  of  gelatin 
the  bouillon  is  first  made  in  the  way  given,  except 
that  its  reaction  is  corrected  after  the  gelatin  has 
been  completely  dissolved,  which  occurs  very  rapidly 
in  hot  bouillon.  The  reaction  of  the  gelatin  of  com- 
merce is  frequently  quite  acid,  so  that  a  much  larger 
amount  of  alkali  is  needed  for  its  neutralization  than 
for  other  media.  It  is  possible,  however,  to  obtain 
from  the  makers  an  excellent  grade  of  gelatin  from 


96  BA  CTERIOL  OGY. 

which  all  acid  has  been  carefully  washed.1  The  gelatin 
is  added  in  the  proportion  of  10  to  12  per  cent.  Its 
complete  solution  may  be  accomplished  either  over  a 
water-bath,  in  the  steam  sterilizer,  or  over  a  free  flame. 
If  the  latter  method  be  practised,  care  must  be  taken 
that  the  mixture  is  constantly  stirred  to  prevent  burn- 
ing at  the  bottom  and  consequent  breaking  of  the  flask, 
if  a  flask  is  employed. 

For  som'e  time  it  has  been  our  practice  to  use  enam- 
elled iron  saucepans,  instead  of  glass  flasks,  for  the 
purpose  of  making  both  gelatin  and  agar-agar ;  by  this 
means  the  free  flame  may  be  employed  without  danger 
of  breaking  the  vessel,  and,  with  a  little  care,  without 
burning  the  media.  Under  any  conditions  it  is  better 
to  protect  the  bottom  of  the  vessel  from  the  direct 
action  of  the  flame  by  the  interposition  of  several  layers 
of  wire  gauze,  a  thin  sheet  of  asbestos-board,  or  an  ordi- 
nary cast-iron  stove-plate. 

When  the  gelatin  is  completely  melted  it  may  be 
filtered  through  a  folded  paper  filter  supported  on  an 
ordinary  funnel ;  if  solution  is  complete,  this  should  be 
very  quickly  accomplished. 

For  the  filtration  of  such  substances  as  gelatin  and 
agar-agar  it  is  of  much  importance  to  have  a  properly 
folded  filter.  To  fold  a  filter  correctly,  proceed  as  fol- 
lows :  a  circular  piece  of  filter  paper  is  folded  exactly 
through  its  centre,  forming  the  fold  1,1'  (Fig.  16) ;  the 
end  1  is  then  folded  over  to  lr,  forming  the  fold  5 ;  1 
and  1'  are  each  then  brought  to  5,  thus  forming  the 
folds  3  and  7  ;  1  is  then  carried  to  the  point  7,  and  the 
fold  4  is  formed,  and  by  carrying  V  to  3  the  fold  6  is 

1  Hesteberg's  acid-free,  gold-label  gelatin  has  given  us  entire  satis- 
faction in  this  respect. 


NUTRIENT  GELATIN. 


97 


produced ;  and  by  bringing  1  to  3  and  V  to  7  the  folds 
2  and  8  result. 


Thus  far  the  ridges  of  all  folds  are  on  the  side  of  the 
paper  next  to  the  table  on  which  we  are  folding.  The 
paper  is  now  taken  up  and  each  space  between  the  seams 
just  produced  is  to  be  subdivided  by  a  seam  or  fold 
through  its  centre,  as  indicated  by  the  dotted  lines  in 
Fig.  16,  but  with  the  creases  on  the  side  opposite  to  that 

FIG.  17. 


occupied  by  the  creases  1,  2,  3,  4,  etc.,  first  made..  As 
each  of  these  folds  is  made  the  paper  is  gradually  folded 
into  a  wedge-shaped  bundle  (Fig.  17,  a),  which  when 
opened  assumes  the  form  of  a  properly  folded  filter 
(seen  in  6,  Fig.  17).  Before  placing  it  upon  the  fun- 
nel it  is  well  to  go  over  each  crease  and  see  that  it  is 
as  tightly  folded  as  possible,  without  tearing  it.  The 

7 


98  BACTERIOLOGY. 

advantage  of  the  folded  filter  is  that  by  its  use  a  much 
greater  filtering  surface  is  obtained,  as  it  is  in  contact  with 
the  funnel  only  at  the  points  formed  by  the  ridges,  leav- 
ing the  greater  part  of  the  flat  surface  free  for  filtration. 

The  employment  of  the  hot-water  funnel,  so  often 
recommended,  has  been  dispensed  with  in  this  work  to 
a  very  large  extent,  for  the  reason  that  if  solution  of 
the  gelatin  is  complete,  filtration  is  so  rapid  as  not  to 
necessitate  the  use  of  an  apparatus  for  maintaining  a 
high  temperature.  The  temperature  at  which  the  hot- 
water  funnel  retains  the  gelatin  is  so  high  that  evapora- 
tion and  concentration  rapidly  occur,  and  in  consequence 
filtration  is,  as  a  rule,  retarded.  The  filtration  is  fre- 
quently done  in  the  steam  sterilizer ;  but  this  too  is 
unnecessary  if  the  gelatin  is  quite  dissolved.  At  the 
ordinary  temperature  of  the  room,  and  by  the  means 
commonly  employed  for  the  filtration  of  other  sub- 
stances, both  gelatin  and  agar-agar  may  be  rapidly 
filtered  if  they  are  completely  dissolved. 

It  not  infrequently  occurs  that,  even  under  the  most 
careful  treatment,  the  filtered  gelatin  is  not  perfectly 
transparent  (the  condition  to  which  it  must  be  brought, 
otherwise  it  is  useless),  and  clarification  becomes  neces- 
sary. For  this  purpose  the  mass  must  be  redissolved, 
and  when  at  a  temperature  between  60°  and  70°  C.  an 
egg,  which  has  been  beaten  up  with  about  50  c.c.  of 
water,  is  added.  The  whole  is  then  thoroughly  mixed 
together  and  again  brought  to  the  boiling-point,  and 
kept  there  until  coagulation  of  the  albumin  occurs.  The 
albumin  coagulates  as  large  flocculent  masses,  and  it  is 
better  not  to  break  them  up  if  it  can  be  avoided,  as 
when  broken  up  into  fine  flakes  they  clog  the  filter  and 
materially  retard  filtration. 


NUTRIENT  GELATIN.  99 

The  practice  sometimes  recommended  of  removing 
these  albuminous  coagula  by  first  filtering  the  gelatin 
through  a  cloth,  and  then  through  paper,  is  not  only 
superfluous,  but  in  most  instances  renders  the  process 
of  filtration  much  more  difficult,  because  of  the  disin- 
tegration of  the  masses  into  finer  particles,  which  have 
the  effect  just  mentioned,  viz.,  of  clogging  the  filter. 

Under  no  circumstances  should  a  filter  be  used  with- 
out first  having  been  moistened  with  water.  If  this  is  not 
done,  the  pores  of  the  paper,  which  are  relatively  large 
when  in  a  dry  state,  when  moistened  by  the  gelatin  not 
only  diminish  in  size,  but  in  contracting  are  often  en- 
tirely occluded  by  the  finer  albuminous  flakes  which 
become  fixed  within  them,  and  filtration  practically 
ceases.  The  preliminary  moistening  with  water  causes 
diminution  of  the  size  of  the  pores  to  such  an  extent 
that  the  finer  particles  of  the  precipitate  rest  on  the 
surface  of  the  paper,  instead  of  becoming  fixed  in  its 
mote* 

During  boiling  it  is  well  to  filter,  from  time  to  time, 
a  few  cubic  centimetres  of  the  gelatin  into  a  test-tube 
and  boil  it  over  a  free  flame  for  a  minute  or  so  ;  in  this 
way  one  can  detect  if  all  the  albumin  has  been  coagu- 
lated— {.  e.j  if  the  solution  is  ready  for  filtration. 

Gelatin  should  not,  as  a  rule,  be  boiled  more  than 
ten  or  fifteen  minutes  at  one  time,  or  be  left  in  the 
steam  sterilizer  for  more  than  thirty  minutes ;  other- 
wise its  property  of  solidifying  may  be  impaired. 

As  soon  as  the  preparation  of  the  gelatin  is  complete, 
whether  it  is  retained  in  the  flask  into  which  it  has 
been  filtered  or  decanted  into  sterilized  test-tubes,  it 
should  be  sterilized  in  the  steam  sterilizer  on  three 
successive  days,  for  fifteen  minutes  each  day,  the  mouth 


100  BACTERIOLOGY. 

of  the  flask  or  the  test-tubes  containing  it  having  been 
previously  closed  with  cotton  plugs. 

NUTRIENT  AGAR-AGAR. — The  preparation  of  nutrient 
agar-agar  by  the  beginner  is  far  too  frequently  a  tedious 
and  time-consuming  operation.  This  is  due  mainly  to 
lack  of  patience  and  to  deviation  from  the  rules  laid 
down  for  the  preparation  of  this  medium.  If  the 
directions  given  below  for  the  preparation  of  nutrient 
agar-agar  be  strictly  observed,  no  difficulty  whatever 
should  be  encountered.  Many  methods  are  recom- 
mended for  its  preparation,  almost  every  worker  having 
some  slight  modification  of  his  own. 

The  methods  that  have  given  us  the  best  results,  and 
from  which  we  have  no  good  grounds  for  departing, 
are  as  follows : 

Prepare  the  bouillon  in  the  usual  way.  Agar-agar 
reacts  neutral  or  very  slightly  alkaline,  so  that  the 
bouillon  may  be  neutralized  before  the  agar-agar  is 
added.  Then  add  finely  chopped  or  powdered  agar- 
agar  in  the  proportion  of  1  to  1.5  per  cent.  Place  the 
mixture  in  a  porcelain-lined  iron  vessel,  and  on  the  side 
of  the  vessel  make  a  mark  at  the  height  at  which  the  level 
of  the  fluid  stands ;  if  a  litre  of  medium  is  being  made, 
add  about  250  to  300  c.c.  more  of  water  and  allow  the 
mass  to  boil  slowly,  occasionally  stirring,  over  a  free 
flame,  from  one  and  a  half  to  two  hours ;  or  until 
the  excess  of  water — i.  e.y  the  250  or  300  c.c.  that 
were  added — has  evaporated.  Care  must  be  taken 
that  the  mixture  does  not  boil  over  the  sides  of  the 
vessel.  From  time  to  time  observe  if  the  fluid  has 
fallen  below  its  original  level ;  if  it  has,  add  water 
until  its  volume  of  1  litre  is  restored.  At  the  end  of 
the  time  given  remove  the  flame  and  place  the  vessel 


NUTRIENT  AGAR-AGAR.  101 

containing  the  mixture  in  a  large  dish  of  cold  water ; 
stir  the  agar-agar  continuously  until  it  has  cooled  to 
about  68°— 70°  C.,  and  then  add  the  white  of  one  egg 
which  has  been  beaten  up  in  about  50  c.c.  of  water ;  or 
the  ordinary  dried  albumin  of  commerce  may  be  dis- 
solved in  cold  water  in  the  proportion  of  about  10  per 
cent,  and  used  ;  the  results  are  equally  as  good  as  when 
eggs  are  employed.  Mix  this  carefully  throughout  the 
agar-agar  and  allow  the  mass  to  boil  slowly  for  about 
another  half-hour,  observing  all  the  while  the  level  of 
the  fluid,  which  should  not  fall  below  the  litre  mark. 
It  is  necessary  to  reduce  the  temperature  of  the  mass 
to  the  point  given,  68°-70°  C. ;  otherwise  the  coagula- 
tion of  the  albumin  will  occur  suddenly  in  lumps  and 
masses  as  soon  as  it  is  added,  and  its  clearing  action 
will  not  be  uniform.  The  process  of  clarification 
with  the  egg  is  purely  mechanical ;  the  finer  particles, 
which  would  otherwise  pass  through  the  pores  of  the 
filter,  being  taken  up  by  the  albumin  as  it  coagulates 
and  being  retained  in  the  coagula. 

At  the  end  of  one-half  hour  the  boiling  mass  may 
be  easily  and  quickly  filtered  through  a  heavy,  folded 
paper  filter  at  the  room-temperature;  as  a  rule,  the 
filtrate  is  as  clear  and  transparent  as  agar-agar  usually 
appears. 

It  may  be  well  to  emphasize  the  fact  that  for  the 
filtration  of  agar-agar  a  hot- water  funnel,  or  any  other 
special  device  for  maintaining  the  temperature  of  the 
mass,  is  entirely  unnecessary.  Agar-agar  prepared  after 
the  methods  just  given  should  pass  through  a  properly 
folded  paper  filter  at  the  rate  of  a  litre  in  from  twelve 
to  fifteen  minutes. 

Another  plan  that  insures  complete  solution  of  the 


102  BACTERIOLOGY. 

agar-agar  without  causing  the  precipitates  often  seen 
when  all  the  ingredients  are  added  at  once  and 
boiled  for  a  long  time,  is  to  weigh  out  the  necessary 
amount  of  agar-agar,  10  or  15  grammes,  and  place  this 
in  1300  or  1400  c.c.  of  water  and  boil  down  over  a  free 
flame  to  1000  c.c.  The  peptone,  salt,  and  beef-extract 
are  then  added  and  the  boiling  continued  until  they 
are  dissolved.  The  clarification  with  egg-albumin 
may  then  be  done,  and  usually  the  mass  filters  quite 
clear  and  does  not  show  the  presence  of  precipitates 
upon  cooling.  If  the  mixture  is  positively  alkaline,  it 
is  not  only  cloudy,  but  it  filters  with  difficulty  ;  if  it  is 
acid,  it  is  usually  quite  clear,  and  filters  more  quickly, 
but,  as  Schultz  has  pointed  out,  it  loses  at  the  same  time 
some  of  its  gelatinizing  properties.  The  bouillon  should 
always  be  neutralized  before  the  agar-agar  is  added  to 
it ;  for  if  the  bouillon  be  acid  from  the  juices  of  the 
meat,  it  robs  the  agar-agar,  under  the  influence  of 
heat,  of  part  of  its  gelatinizing  power,  which  will  not 
be  regained  by  subsequent  neutralization. 

Another  method  by  which  agar-agar  can  be  easily 
and  quickly  melted  is  by  steam  under  pressure.  If 
the  flask  containing  the  mixture  of  bouillon  and  agar- 
agar  be  kept  in  the  digester  or  autoclave  for  ten  minutes 
with  the  steam  under  a  pressure  of  about  one  atmos- 
phere, as  shown  by  the  gauge,  the  agar-agar  will  be 
found  at  the  end  of  this  time  completely  melted,  and  fil- 
tration may  then  be  accomplished  with  but  little  difficulty. 

If  glycerin  is  to  be  added  to  the  agar-agar,  it  is  done 
after  filtration  and  before  sterilization.  The  nutritive 
properties  of  the  media  for  certain  organisms,  particu- 
larly the  tubercle  bacillus,  are  increased  by  the  addition 
of  glycerin  in  the  proportion  of  5  to  7  per  cent. 


PREPARATION  OF  POTATOES.  103 

If  after  filtration  a  fine  flocculent  precipitate  is  seen, 
look  to  the  reaction  of  the  medium.  If  it  is  quite 
alkaline,  neutralize,  boil,  and  filter  again.  If  the 
reaction  is  neutral  or  only  very  slightly  acid,  dissolve 
and  again  clarify  with  egg-albumin  by  the  method 
given. 

The  most  important  feature  of  all  the  media,  aside 
from  the  correct  proportion  of  the  ingredients,  is  their 
reaction.  They  must  be  neutral  or  very  slightly  alkaline 
to  litmus.  (See  remarks  on  Neutralization  of  Media.) 
Only  a  few  organisms  develop  well  on  media  of  an  acid 
reaction.  In  all  of  the  media  mentioned  above  the  meat- 
ex  tracts  now  on  the  market  may  usually  be  substituted 
for  the  meat  itself  in  preparing  the  bouillon.  They 
may  be  employed  in  the  proportion  of  from  two  to  four 
grammes  to  the  litre  of  water. 

PREPARATION  OF  POTATOES. — Potatoes  are  prepared 
for  use  in  two  ways : 

1.  They  are  taken  as  they  come  to  market — old 
potatoes  being  usually  recommended — and  carefully 
scrubbed  under  a  water-tap  with  a  stiff*  brush  until 
all  adherent  dirt  has  been  removed;  "the  eyes"  and 
all  discolored  or  decayed  parts  are  carefully  removed 
with  a  pointed  knife.  They  are  then  placed  in  a  solu- 
tion of  corrosive  sublimate  of  the  strength  of  1  : 1000, 
where  they  are  allowed  to  remain  for  twenty  minutes ; 
at  the  end  of  this  time,  without  rinsing  off  the  sublimate, 
they  are  placed  in  a  covered  tin  bucket  with  a  perforated 
bottom  and  sterilized  in  the  steam  sterilizer  for  forty- 
five  minutes.  On  the  second  and  third  days  the  steril- 
ization is  repeated  for  fifteen  to  twenty  minutes  each  day. 
They  must  not  be  removed  from  the  sterilizing  bucket 
until  sterilization  is  complete,  when  they  are  ready  for 


104  BACTERIOLOGY. 

use.  When  prepared  in  this  way  they  are  usually  in- 
tended to  be  cut  in  half,  and  the  cultivation  of  the 
organisms  is  to  be  conducted  upon  the  flat  surfaces 
of  the  sections.  (Koch's  original  method.) 

This  method  requires  some  care  to  prevent  contam- 
ination during  manipulation.  The  hand  which  is  to 
take  up  the  potato  from  the  bucket,  which  until  now  has 
remained  covered,  is  first  disinfected  in  the  sublimate 
solution  for  ten  minutes ;  the  potato  is  then  taken  up 
between  the  thumb  and  index  finger  and  severed  in 
two  by  a  knife  which  has  just  been  sterilized  in  a  free 
flame  until  it  is  quite  hot.  The  knife  is  passed  not 
quite  through  the  potato,  but  nearly  so.  A  large  glass 
culture-dish  for  the  reception  of  the  halves  of  the 
potato,  having  been  disinfected  for  twenty  minutes  with 
1  : 1000  sublimate  solution  and  then  drained  of  all  ad- 
herent solution,  should  be  at  hand  ready  for  the  potato ; 
the  cover  is  removed,  and  by  twisting  the  knife  gently 
the  halves  of  the  potato  may  be  caused  to  fall  apart  in 
the  dish  and  usually  to  fall  upon  their  convex  surfaces, 
leaving  the  flat  sections  uppermost.  The  cover  of 
the  dish  is  replaced  and  the  potatoes  are  ready  for 
inoculation. 

2.  Preparation  of  potatoes  for  test-tube  cultures.  Method 
of  Bolton.1  If  the  potatoes  are  to  be  employed  for  test- 
tube  cultures,  one  simply  scrubs  off  the  coarser  particles 
of  dirt  with  water  and  a  brush,  and  with  a  cork- borer 
punches  out  cylindrical  bits  of  potato  which  will  fit 
loosely  into  the  test-tubes  to  be  used.  On  each  bit  of 
potato  is  then  to  be  cut  a  slanting  surface  running  from 
about  the  junction  of  the  first  and  second  thirds  of  the 
cylinder  to  the  diagonally  opposite  end.  These  cylin- 
1  Medical  News,  vol.  i.  of  1887,  No.  12,  p.  318. 


PREPARATION  OF  POTATOES. 


105 


FIG.  18. 


ders  of  potato  are  to  be  left  in  running  water  over 
night,  otherwise  they  will  be  very  much  discolored  by  the 
sterilization  to  which  they  are  to  be  subjected.  After 
being  thus  washed  they  are  placed  in  pre- 
viously prepared  test-tubes,  one  piece  in  each 
tube,  with  the  slanting  surface  up,  the  cot- 
ton plugs  of  the  tubes  replaced,  and  they 
are  then  to  be  sterilized  in  steam  for  fifteen 
to  twenty  minutes  on  each  of  three  successive 
days.  Or  the  entire  sterilization  may  be  ac- 
complished in  the  autoclave,  with  the  steam 
under  a  pressure  of  one  atmosphere,  by  a 
single  exposure  of  twenty  to  twenty-five 
minutes.  Potatoes  thus  prepared  have  the 
appearance  seen  in  Fig.  18,  except  that  there 
is  no  growth  upon  the  surface  as  is  shown 
in  the  cut. 

For  some  purposes  potatoes  may  be  ad- 
vantageously peeled,  sliced  into  discs  of 
about  1  cm.  in  thickness,  and  placed  in 
small  glass  dishes  provided  with  covers, 
similar  to  the  ordinary  Petri  dishes.  The 
dish  and  its  contents  are  then  sterilized 
by  steam  in  the  usual  way  (method  suggested  by  von 
Esmarch).  By  this  plan  a  relatively  large  area  for 
cultivation  is  obtained. 

Potatoes  may  also  be  boiled,  or  steamed,  and  mashed, 
and  the  mass  placed  in  covered  dishes,  test-tubes,  or 
flasks,  and  sterilized.  By  this  method  one  obtains  in 
the  mass  a  mean  of  the  composition  of  the  several  pota- 
toes, or  bits  of  potatoes,  used  in  making  it,  an  advan- 
tage where  uniformity  is  desired. 

Care  must  be  given  to  the  sterilization  of  potatoes, 


Potato  in  test- 
tube. 


106  BACTERIOLOGY. 

because  they  always  have  adhering  to  them  the  organ- 
isms commonly  found  in  the  ground,  the  spores  of  which 
are  among  the  most  resistant  known.  The  so-called 
potato  bacillus  is  one  of  this  group ;  it  is  an  organism 
which  is  not  infrequently  more  or  less  of  an  obstacle 
to  the  work  of  the  beginner. 

BLOOD-SERUM. — By  the  original  method  of  preparing 
blood-serum  a  great  many  precautions  were  taken  that 
have  been  found  unnecessary  to  the  success  of  the  more 
modern  plans. 

It  is  possible  to  collect  serum  from  small  animals  and 
in  small  quantities  under  such  precautions  that  it  is  per- 
haps not  contaminated ;  but,  ordinarily,  for  laboratory 
purposes  a  larger  quantity  is  needed,  so  that  slaughter- 
houses are  the  source  from  which  it  is  usually  obtained, 
and  here  a  certain  amount  of  contamination  is  unavoid- 
able, though  its  extent  may  be  limited  by  proper  pre- 
cautions. 

The  steps  formerly  thought  essential  to  the  collection 
of  blood  and  the  preparation  of  serum  for  culture  pur- 
poses were  about  as  follows : 

The  animal  from  which  the  blood  is  to  be  collected 
should  be  drawn  up  to  the  ceiling  by  the  hind  legs  ;  the 
head  should  be  held  well  back,  and  with  one  pass  of  a 
very  sharp  knife  the  throat  should  be  completely  cut 
through.  The  blood  which  spurts  from  the  severed  ves- 
sels should  be  collected  in  large  glass  jars  which  have 
been  previously  cleaned  and  disinfected,  and  all  traces  of 
the  disinfectant  removed  with  alcohol  and,  finally,  with 
ether.  The  latter  evaporates  very  quickly  and  leaves 
the  jar  quite  dry.  The  jars  should  be  provided  with 
covers,  which  close  hermetically ;  these,  too,  should  be 
carefully  disinfected.  The  best  form  of  vessels  for 


BLOOD-SERUM.  107 

the  purpose  is  the  large  museum-jar  of  about  one 
gallon  capacity,  which  closes  by  a  cover  that  can  be 
tightly  screwed  down  upon  a  rubber  joint.  From  two 
such  jarfuls  of  blood  one  can  recover  quite  a  large  quan- 
tity of  clear  serum,  ordinarily  from  500  to  700  c.c.  The 
jars  having  been  filled  with  blood,  their  covers  are  placed 
loosely  upon  them  and  they  are  allowed  to  stand  for 
about  fifteen  minutes  until  clotting  has  begun.  At  the 
end  of  this  time  a  clean  glass  rod  is  passed  around  the 
edges  of  the  surface  of  the  clot  to  break  up  any  adhe- 
sions to  the  side  of  the  jar  that  may  have  formed,  and 
which  would  prevent  the  sinking  of  the  clot  to  the 
bottom.  The  covers  are  then  replaced  and  tightly 
clamped  in  position,  and  with  as  little  agitation  as  pos- 
sible the  jars  are  placed  in  an  ice-chest,  where  they 
remain  for  twenty-four  to  forty-eight  hours.  The 
temperature  should,  however,  not  be  low  enough  to 
prevent  coagulation,  but  should  be  sufficiently  low  to 
interfere  with  the  development  of  any  living  organ- 
isms that  may  be  present.  The  temperature  of  the 
ordinary  domestic  refrigerator  is  sufficient  for  the 
purpose.  After  twenty-four  to  forty-eight  hours  the 
clot  will  have  become  firm,  and  will  be  seen  at  the 
bottom  of  the  jar.  Above  it  is  a  quantity  of  dark 
straw-colored  serum.  The  serum  may  then  be  drawn 
off*  with  a  sterilized  pipette  and  placed  in  tall  cylinders 
that  have  previously  been  plugged  with  cotton  wadding 
and  sterilized.  After  treating  all  the  serum  in  this  way, 
care  having  been  taken  to  exclude  as  much  as  possible  of 
the  coloring-matter  of  the  blood,  it  may  be  placed  again 
in  the  ice-chest  for  twenty-four  hours,  during  which 
time  the  corpuscular  elements  will  sink  to  the  bottom, 
leaving  the  supernatant  fluid  quite  clear.  This  may 


108  BACTERIOLOGY. 

then  be  pipetted  off,  either  into  sterilized  test-tubes, 
about  8  c.c.  to  each  tube,  or  into  small  sterilized  flasks 
of  about  100  c.c.  capacity.  It  is  then  to  be  sterilized 
by  the  intermittent  method  at  low  temperatures,  viz.,  for 
one  hour  on  each  of  five  consecutive  days  at  a  tempera- 
ture of  68°-70°  C.  During  the  intervening  days  it  is 
to  be  kept  at  the  room-temperature  to  permit  of  the 
development  of  any  spores  that  may  be  present  into 
their  vegetative  forms,  in  which  condition  they  are 
killed  by  an  hour's  exposure  to  the  temperature  of 
70°  C. 

FIG.  19. 


Chamber  for  sterilizing  and  solidifying  blood-serum.     (Kocn. ) 

At  the  end  of  this  time  the  serum  in  the  tubes  may 
either  be  retained  as  fluid  serum  or  solidified  at  between 
76°-80°  C.  In  solidifying  the  serum  the  tubes  should 
be  placed  in  an  inclined  position,  so  that  as  great  a  sur- 
face as  possible  may  be  given  to  the  serum.  The  proc- 
ess of  solidification  requires  constant  attention  if  good 


BLOOD-SERUM.  109 

results  are  to  be  obtained — i.  e.,  if  a  translucent,  solid 
medium  is  the  result.  If  the  old,  small  form  of  appa- 
ratus be  employed  (Fig.  19),  the  solidification  can  be 
accomplished  in  a  shorter  time  than  if  the  larger 
forms  commonly  employed  are  used.  No  definite 
rule  for  the  time  that  will  be  required  can  be  laid 
down,  for  this  is  not  constant.  If  the  small  solidify- 
ing apparatus  be  used,  very  good  results  may  be  ob- 
tained in  about  two  hours  at  78°  C.  It  frequently 
requires  a  longer  time  at  a  higher  temperature  than 
has  been  mentioned.  This  is  especially  the  case  with 
Loffler's  serum-mixture. 

The  best  results  are  obtained  when  a  low  temperature 
is  employed  for  a  long  time.  Under  any  circumstances 
the  tubes  should  be  observed  from  time  to  time  through 
the  glass  door  or  cover  with  which  the  solidifying  oven 
is  provided,  and  each  time  the  oven  should  be  slightly 
jarred  with  the  hand  to  see  if  solidification,  as  indi- 
cated by  the  disappearance  of  tremors  from  the  serum, 
is  beginning.  If  the  temperature  gets  too  high,  or  the 
exposure  is  too  long,  an  opaque  medium  results.  The 
temperature  to  be  observed  is  that  of  the  air  inside 
the  chamber,  and  also  that  of  the  water  surrounding  it. 
The  latter  is  usually  a  degree  or  two  higher  than  the 
former.  The  tubes  should  not  rest  directly  upon  the 
heated  bottom  or  against  the  heated  sides  of  the  cham- 
ber, but  should  lie  upon  racks  of  wood  or  wire,  and  be 
protected  from  the  sides  by  a  wire  screen  or  gauze  :  in 
this  way  all  the  tubes  are  exposed  to  about  the  same 
temperature.  The  thermometer  which  indicates  the 
temperature  inside  the  chamber  should  not  touch  the 
surfaces,  but  should  either  be  suspended  free  from 
above  through  a  cork  in  the  top  of  the  apparatus,  if 


110  BACTERIOLOGY. 

a  large  form  of  apparatus  be  used  ;  or  should  lie  upon 
a  rack  of  cork  or  wood,  its  bulb  being  free  and  a  little 
lower  than  the  other  extremity,  if  the  small,  old-fash- 
ioned apparatus  of  Koch  be  employed.  The  latter  form 
is  preferable,  as  it  is  more  easily  managed. 

When  solidification  is  complete  the  tubes  are  to  be 
retained  in  the  erect  position,  and,  unless  they  are 
intended  for  immediate  use,  must  be  prevented  from 
drying.  The  superfluous  ends  of  the  cotton  plugs 
should  be  burned  off,  and  the  mouths  of  the  tubes  may 
then  be  covered  by  sterilized  rubber  caps,  or,  as  Ghris- 
key  suggests,  they  may  be  closed  with  sterilized  corks 
pushed  in  on  top  of  the  cotton  plugs.  Even  with  the 
greatest  care  it  not  uncommonly  happens  that  one  or 
two  of  a  lot  of  tubes  thus  prepared  and  protected  will 
become  contaminated.  This  is  usually  due  to  spores  of 
moulds  that  have  fallen  into  the  rubber  caps  or  on  the 
cotton  plugs  during  manipulation,  and,  finding  no 
means  of  outward  growth,  have  thrown  their  hyphse 
downward  through  the  cotton  into  the  tube,  and  their 
spores  have  fallen  on  the  surface  of  the  serum  and 
developed  there. 

The  foregoing  is,  in  the  main,  the  plan  originally 
recommended  by  Koch  for  the  preparation  of  this 
medium.  In  recent  times,  however,  particularly  since 
the  diagnosis  of  diphtheria  by  the  method  of  Loffler  has 
become  so  general,  and  large  quantities  of  serum-tubes 
have  been  found  necessary,  a  modification  has  been 
suggested  that  has,  in  this  country  at  least,  almost  en- 
tirely supplanted  the  method  of  Koch.  The  popularity 
of  the  Councilman-Mallory  method  is  due  to  the  fol- 
lowing facts :  by  it  the  serum  is  more  quickly  and 
easily  prepared ;  rigid  precautions  against  contamination 


BLOOD-SERUM.  Ill 

during  collection  of  the  serum  are  not  so  necessary  ;  and 
the  resulting  medium,  while  not  transparent  or  even 
translucent  (points  aimed  at  in  the  original  method), 
fully  meets  all  the  requirements. 

The  special  points  in  the  method  are :  the  serum  is 
decanted  into  test-tubes  as  soon  as  obtained;  it  is  then 
firmly  coagulated  in  a  slanting  position  in  the  dry-air 
sterilizer  at  from  80°  to  90°  C. ;  it  is  then  sterilized  in 
the  steam  sterilizer  at  100°  C.  on  three  successive  days, 
as  in  the  case  of  other  culture-media.  It  may  then  be 
protected  against  evaporation  by  sterilized  rubber  caps 
or  sterilized  corks  in  the  way  already  described,  and  set 
aside  until  needed. 

Unless  the  coagulation  in  the  dry  sterilizer  be  com- 
plete, the  surface  of  the  serum  will  be  found  to  be  blis- 
tered and  pitted  by  bubbles  and  cavities  after  it  has 
been  subjected  to  the  steam  sterilization.  A  similar 
formation  of  cavities  over  the  surface  of  the  serum  will 
occur  if  the  temperature  of  the  hot-air  sterilizer,  in 
which  it  is  solidified,  is  allowed  to  get  above  90°  C., 
or  if  it  be  elevated  to  this  point  too  quickly. 

It  is  of  no  special  advantage  to  have  the  serum  clear, 
as  the  admixture  of  blood-coloring-matter  does  not  affect 
its  nutritive  properties. 

It  is  often  desirable  to  obtain  small  quantities  of  blood- 
scrum  under  strictly  aseptic  precautions,  and  for  this  pur- 
pose Xuttall  (Centralb.fdr  Bakt.  u  Parasitenkunde,  1892, 
Bd.  xi.  p.  539)  suggests  a  very  convenient  method.  By 
the  use  of  a  sterilized  vessel,  of  the  shape  shown  in  Fig. 
20,  from  10  to  100  c.c.  of  blood  can  be  collected,  and  if 
proper  precautions  are  observed  no  contamination  by 
bacteria  need  occur.  The  collecting  bulb  is  used  in  the 
following  way :  an  artery,  either  femoral  or  carotid,  is 


112  BACTERIOLOGY. 

exposed,  and  around  it  two  ligatures  are  placed  ;  that 
distant  from  the  heart  is  tightened,  while  the  one  near- 
est the  heart  is  left  loose ;  between  the  latter  and  the 
heart  the  artery  is  clamped.  A  small  slit  is  then  made 
in  its  wall,  into  which  the  point  a  of  the  bulb  is  intro- 
duced and  the  artery  bound  tightly  around  it  with  the 

Fia.  20. 


a 

Nuttall's  bulb  for  collecting  blood-serum  under  antiseptic  precautions. 

hitherto  loose  ligature  ;  the  clamp  is  removed  and  the 
bulb  quickly  fills  with  blood.  The  clamp  is  now  again 
put  in  position,  the  point  of  the  bulb  removed  and 
sealed  in  a  gas-flame,  the  loose  ligature  tightened, 
the  wound  closed,  and  the  bulb  containing  the  blood  is 
set  aside  in  a  cool  place  until  coagulation  has  occurred. 
The  serum  is  most  easily  withdrawn  from  the  bulb  by 
means  of  a  pipette,  closed  above  with  a  cotton-plug,  and 
supplied  with  a  piece  of  rubber-tubing  about  one-half 
metre  long,  with  glass  mouth-piece.  By  holding  the 
pipette  in  the  hand  and  sucking  upon  the  rubber  tube 
one  can  more  easily  direct  the  point  of  the  pipette  than 


SPECIAL  MEDIA.  113 

if  it  is  used  in  the  ordinary  way.  The  bulbs  are  easily 
blown,  and  after  having  been  sealed  at  the  point  and 
plugged  with  cotton  can  be  kept  on  hand  just  as  are 
sterilized  test-tubes. 

It  is  sometimes  desirable  to  preserve  blood-serum  in 
a  fluid  state.  This  can  be  done  by  the  fractional  method 
of  sterilization  at  low  temperatures,  already  described, 
or  with  much  less  effort,  and  without  the  use  of  heat, 
by  a  method  that  we  have  found  very  satisfactory.  In 
the  course  of  Kirschner's  investigations  chloroform  was 
shown  to  possess  decided  disinfectant  properties ;  as  it  is 
quite  volatile,  it  is  easily  got  rid  of  when  its  disinfectant 
or  antiseptic  properties  are  no  longer  required.  If,  there- 
fore, the  serum  to  be  preserved  be  placed  in  a  closely 
stoppered  flask  and  enough  chloroform  added  to  form  a 
thin  layer,  about  2  mm.,  on  the  bottom,  the  serum  may 
be  kept  indefinitely  without  contamination,  so  long  as 
the  chloroform  is  not  permitted  to  evaporate.  When 
required  for  use  the  serum  is  decanted  into  test-tubes, 
which  are  then  placed  in  a  water-bath  at  about  50°  C. 
until  all  the  chloroform  has  been  driven  off;  this  can 
be  determined  by  the  absence  of  its  characteristic  odor. 
The  serum  may  then  be  solidified,  sterilized  by  heat, 
and  employed  for  culture  purposes.  We  have  found 
serum  so  preserved  to  answer  all  requirements  as  a  cul- 
ture-medium. 

SPECIAL,  MEDIA. — The  media  just  described — bou- 
illon, nutrient  gelatin,  nutrient  agar-agar,  potato,  and 
blood-serum — are  those  in  general  use  in  the  laboratory 
for  purposes  of  isolation  and  study  of  the  ordinary 
forms  of  bacteria.  For  the  finer  points  of  differentia- 
tion special  media  have  been  suggested ;  a  few  of  them 
will  be  mentioned. 


114  BACTERIOLOGY. 

Milk.  Fresh  milk  should  be  allowed  to  stand  over 
night  in  an  ice-chest,  the  cream  then  removed,  and  the 
remainder  of  the  milk  pipetted  into  test-tubes,  about 
8  c.c.  to  each  tube,  and  sterilized  by  the  intermittent 
process,  at  the  temperature  of  steam,  for  three  succes- 
sive days. 

The  separation  of  the  cream  may  be  accelerated  and 
rendered  more  complete  if  the  cylinder  containing  the 
milk  be  placed  in  the  steam  sterilizer  for  fifteen  minutes 
before  it  is  placed  in  the  ice-chest. 

The  cream  is  best  separated  from  the  milk  by  the  use 
of  a  cylindrical  vessel  with  a  stopcock  at  the  bottom,  by 
means  of  which  the  milk,  devoid  of  cream,  may  be 
drawn  off.  A  Chevalier  creamometer  with  a  stopcock 
at  the  bottom  serves  the  purpose  very  well.  It  should 
be  covered  while  standing.1 

Milk  may  be  used  as  a  culture-medium  without  any 
addition  to  it,  or,  before  sterilizing,  a  few  drops  of 
litmus  tincture  may  be  added,  just  enough  to  give  it  a 
pale-blue  color.  By  this  means  it  will  be  seen  that 
different  organisms  bring  about  different  reactions  in 
the  medium  :  some  producing  alkalies,  which  cause  the 
blue  color  to  be  intensified ;  others  producing  acids,  which 
change  it  to  red ;  while  others  bring  about  neither  of 
these  changes.  Similarly  litmus  solution  is  often  added 
to  gelatin  and  agar-agar  for  the  same  purpose. 

Milk  may  also  be  employed  as  a  solid  culture-medium 
by  the  addition  to  it  of  gelatin  or  agar-agar  in  the  pro- 
portions given  for  the  preparation  of  ordinary  nutri- 
ent gelatin  or  agar-agar.  It  has,  however,  in  this  form 

1  For  some  time  past  we  have  been  using  what  is  technically  known 
as  "  separator  milk  " — i.  e.,  the  fluid  left  after  milk  has  been  deprived 
of  its  fat  (cream)  by  centrifugal  force. 


SPECIAL  MEDIA.  115 

tin  disadvantage  of  not  being  transparent,  and  can 
therefore  best  be  used  for  the  study  of  those  organisms 
which  grow  upon  the  surface  of  the  medium  without 
causing  liquefaction. 

Nutrient  gelatin  and  agar-agar  can  also  be  prepared 
from  neutral  milk-whey,  obtained  from  milk  after  pre- 
cipitation of  the  casein. 

Dunham's  peptone  solution.  The  medium  usually 
known  as  Dunham's  solution  is  prepared  according  to 
the  following  formula : 

Dried  peptone  .    .  •».  .   .   -. -  .. 1    part. 

Sodium  chloride 0.5    " 

Distilled  water .: 100    parts. 

It  is  usually  of  a  neutral  or  slightly  alkaline  reac- 
tion, and  neutralization  is  not,  therefore,  necessary. 
It  is  filtered,  decanted  into  tubes  or  flasks,  and  ster- 
ilized in  the  steam  sterilizer  in  the  ordinary  way. 
The  most  common  use  to  which  this  solution  is  put 
is  in  determining  if  the  organism  under  considera- 
tion possesses  the  property  of  producing  indol  as  one 
of  its  metabolic  products.  It  is  essential  for  accu- 
racy that  the  preparation  of  dried  peptone  employed 
should  be  as  nearly  chemically  pure  as  is  possible, 
and  indeed  the  other  ingredients  should  be  corre- 
spondingly free  from  impurities.  Gorini  (Centralbhdt 
fur  Balderiologie  und  Parasitenkunde,  1893,  Bd.  xiii. 
p.  790)  calls  attention  to  the  fact  that  impurities  in 
the  peptone,  particularly  the  presence  of  carbohydrates, 
so  interfere  with  the  production  of  indol  by  certain 
bacteria  that  otherwise  produce  it,  that  it  is  ofttimes 
impossible,  under  such  circumstances,  to  obtain  the 
characteristic  color-reaction  of  this  body,  and  where  it 


116  BACTERIOLOGY. 

is  obtained  it  is  always  after  a  much  longer  time  than  is 
the  case  where  peptone  free  from  these  substances  has 
been  used. 

Peckham  has  also  demonstrated  that  where  bacteria 
have  the  property  of  forming  indol  and  also  of  fer- 
menting carbohydrates,  their  proteolytic  function,  as 
evidenced  by  the  appearance  of  indol  as  a  product 
of  metabolism,  may  be  completely  suppressed  by  the 
addition  of  such  fermentable  carbohydrates  as  glucose, 
saccharose,  and  lactose  to  the  proteid  solution  in  which 
they  are  developing.1 

Gorini  suggests  the  advisability  of  testing  the  purity 
of  all  peptone  preparations  before  using  them,  by  means 
of  the  reaction  that  they  exhibit  with  Fehling's  alka- 
line copper  solution.  Under  the  influence  of  this 
agent  pure  peptone  in  solution  gives  a  violet  color  (the 
biuret  reaction),  which  remains  permanent  even  after 
boiling  for  five  minutes.  If,  instead  of  a  violet  color, 
there  appears  a  red  or  reddish-yellow  precipitate,  the 
peptone  should  be  discarded,  as  in  his  experience  no 
indol  is  produced  from  peptone  giving  this  reaction. 
Both  the  peptone  solution  and  that  of  the  copper  (par- 
ticularly the  latter)  should  be  relatively  dilute  in  order 
for  the  reaction  to  be  successful. 

Peptone  rosolie  acid  solution.  Peptone  solution  con- 
taining rosolic  acid  serves  well  for  the  detection  of  alter- 
ations in  reaction.  It  consists  of  the  peptone  solution 
of  Dunham,  to  each  100  c.c.  of  which  2  c.c.  of  the 
following  solution  are  added  : 

Rosolic  acid  (coralline) 0.5  gramme. 

Alcohol  (80  per  cent.) 100      c.c. 

1  See  Journal  of  Experimental  Medicine,  1897,  vol.  ii.  p.  549. 


SPECIAL  MEDIA.  117 

This  is  to  be  boiled,  filtered,  and  decanted  into  clean, 
sterilized  test-tubes,  about  8  to  10  c.c.  to  each  tube. 
The  tubes  are  then  to  be  sterilized  in  the  usual  way  by 
steam.  When  sterilization  is  completed  and  the  tubes 
cooled  the  solution  will  be  of  a  very  pale  rose  color, 
which  disappears  entirely  under  the  action  of  acids,  but 
becomes  much  more  intense  under  the  influence  of  alka- 
lies. We  have  used  this  solution  for  some  time  for  the 
study  of  the  reactions  produced  by  different  organisms, 
and  find  it  a  valuable  addition  to  our  means  of  differen- 
tiating bacteria. 

Rosolic  acid  cannot  be  used  in  solutions  containing 
glucose,  as  the  reducing  action  of  the  latter  deprives  it 
of  its  color. 

Lactose  litmus-agar,  or  litmus-gelatin  of  Wurtz.  A 
medium  of  much  use  in  the  differentiation  of  bacteria 
is  that  recommended  by  Wurtz,  consisting  of  slightly 
alkaline  nutrient  agar-agar,  to  which  from  2  to  3  per 
cent,  of  lactose  and  sufficient  litmus  tincture  to  give 
it  a  pale-blue  color  have  been  added.  Bacteria  capable 
of  causing  fermentation  of  lactose  when  grown  on  this 
medium  develop  into  colonies  of  a  pale-pink  color  and 
cause,  likewise,  a  reddening  of  the  surrounding  medium, 
owing  to  the  production  of  acid  as  a  result  of  their 
action  upon  the  lactose ;  while  other  bacteria,  incapable 
of  such  fermentative  activities,  grow  as  pale-blue  colonies 
and  cause  no  reddening  of  the  surrounding  medium. 
It  is  especially  useful  in  the  differentiation  of  the  bacillus 
of  typhoid  fever,  which  does  not  possess  the  property 
of  bringing  about  fermentation  of  lactose,  from  other 
organisms  that  simulate  it  in  many  other  respects,  but 
which  do  possess  this  property. 

Its  preparation  is  as  follows :  to  nutrient  agar-agar 


118  BACTERIOLOGY. 

or  gelatin,  the  alkalinity  of  which  is  such  that  1  c.c. 
will  require  0.1  c.c.  of  a  1  :  20  normal  sulphuric-acid 
solution  to  neutralize  it,  lactose  is  added  in  the  propor- 
tion of  2  or  3  per  cent. ;  it  is  then  decanted  into  test- 
tubes  and  "sterilized  in  the  usual  way.  When  ster- 
ilization is  complete  enough  sterilized  litmus  tincture 
should  be  added  to  each  tube  to  give  a  decided,  though 
not  very  intense,  blue  color.  This  must  be  done  care- 
fully, to  avoid  contamination  of  the  tubes  during  ma- 
nipulation. It  is  better  not  to  add  the  litmus  tincture 
before  sterilizing  the  tubes,  as  its  color-characteristics 
are  altered  by  contact  with  organic  matters  under  the 
influence  of  heat.  This  medium  is  used  for  both  test- 
tube  and  plate  cultivation,  just  as  is  ordinary  agar-agar 
and  gelatin. 

Loffler's  blood-serum  mixture.  Loftier' s  blood-serum 
mixture  consists  of  one  part  of  neutral  meat-infusion 
bouillon,  containing  1  per  cent,  of  grape-sugar,  and 
three  parts  of  blood-serum.  This  mixture  is  placed  in 
test-tubes,  sterilized,  and  solidified  in  exactly  the  way 
given  for  blood-serum.  It  requires  for  its  solidification 
a  somewhat  higher  temperature  and  a  longer  exposure 
to  this  temperature  than  does  blood-serum  to  which  no 
bouillon  has  been  added.  (See  also  the  Councilman- 
Mallory  method.) 

Guarniari's  agar-gelatin : 

Meat-infusion 950  c.c. 

Sodium  chloride 5  grammes. 

Peptone 25-30    " 

Gelatin 40-60     " 

Agar-agar 3-4      " 

Water 50  c.c. 

The  special  point  in  the  preparation  of  this  medium 
is  its  reaction,  which  should  be  exactly  neutral. 


SPECIAL  MEDIA.  119 

The  full  list  of  special  media  is  too  extensive  to  be 
given  in  a  book  of  this  size.  For  their  description  the 
reader  is  referred  to  the  current  literature.  Those  that 
have  been  given  above  will  suffice  for  obtaining  a  clear 
understanding  of  the  principles  of  the  subject. 

NOTE. — The  term  "  meat-infusion  "  always  implies  a 
watery  extract  of  meat  made  by  mixing  500  grammes 
of  finely  chopped  lean  meat  and  1  litre  of  water,  and 
allowing  them  to  stand  in  a  cool  place  for  twenty-four 
hours.  At  the  end  of  this  time  the  fluid  portion  is 
strained  off  through  a  coarse  towel,  and  this  is  what  is 
meant  by  the  "  meat-infusion." 


CHAPTER   VI. 

Preparation  of  the  tubes,  flasks,  etc.,  in  which  the  media  are  to  be 
preserved. 

WHILE  the  media  are  in  course  of  preparation  it  is 
well  to  get  the  test-tubes  and  flasks  ready  for  their 
reception,  and  it  is  essential  that  they  should  be  as  clean 
as  it  is  possible  to  make  them.  For  this  purpose  it  is 
advisable  that  both  new  tubes  and  those  which  have 
previously  been  used  should  be  boiled  for  about  thirty 
to  forty-five  minutes  in  a  2  to  3  per  cent,  solution  of 
common  soda ;  it  is  not  necessary  to  be  exact  as  to 
strength,  but  it  should  not  be  weaker  than  this.  At  the 
end  of  this  time  they  are  to  be  carefully  swabbed  out 
with  a  cylindrical  bristle  brush,  preferably  one  with 
a  reed  handle  (Fig.  21,  a),  as  those  with  wire  handles 

FIG.  21. 


Brushes  for  cleaning  test-tubes. 


are  apt  to  break  through  the  bottoms  of  the  tubes, 
though  Messrs.  Lentz  &  Sons,  of  this  city,  have  in 
large  part  eliminated  this  objection  from  the  wire-handle 
brush  depicted  in  Fig.  21,  b.  All  traces  of  adherent 
120 


FILLING   THE  TUBES.  121 

material  should  be  carefully  removed.  When  the  tubes 
are  quite  clean  they  may  be  rinsed  in  a  warm  solution 
of  commercial  hydrochloric  acid  of  the  strength  of 
about  1  per  cent.  This  is  to  remove  the  alkali.  They 
are  then  to  be  thoroughly  rinsed  in  clear,  running  water, 
and  stood  top  down  until  the  water  has  drained  from 
them.  When  dry  they  are  to  be  plugged  with  raw 
cotton ;  this  requires  a  little  practice  before  it  can  be 
properly  done.  The  cotton  should  be  introduced  into 
the  mouths  of  the  tubes  in  such  a  way  that  no  cracks 
or  creases  exist.  The  plug  should  fit  neither  too  tightly 
nor  too  loosely,  but  should  be  just  firmly  enough  in 
position  to  sustain  the  weight  of  the  tube  into  which 
it  is  placed  when  held  up  by  the  portion  which  projects 
from  and  overhangs  the  mouth  of  the  tube.  The  tubes 
thus  plugged  are  now  to  be  placed  upright  in  a  wire 
basket  and  heated  for  one  hour  in  the  hot-air  sterilizer 
at  a  temperature  of  about  150°  C.  A  very  good  guide 
for  this  process  of  sterilization  is  to  observe  the  tubes 
from  time  to  time,  and  as  soon  as  the  cotton  has  become 
a  very  light-brown  color,  not  deeper  than  a  dark-cream 
tint,  to  consider  sterilization  complete.  The  tubes  are 
then  removed  and  allowed  to  cool. 

The  cotton  used  for  this  purpose  should  be  the  ordi- 
nary cotton  batting  of  the  shops,  and  not  absorbent 
cotton ;  the  latter  becomes  too  tightly  packed,  and  is, 
moreover,  much  too  expensive  for  this  purpose. 

Care  should  be  taken  not  to  burn  the  cotton,  other- 
wise the  tubes  will  become  coated  with  a  dark-colored, 
empyreumatic,  oily  deposit,  which  necessitates  recleans- 
ing. 

FILLING  THE  TUBES. — When  the  tubes  are  cold 
they  may  be  filled.  This  is  best  accomplished  by  the 


122 


BACTERIOLOGY. 


use  of  a  separating  funnel,  such  as  is  shown  in  Fig. 
22.  The  liquefied  medium  is  poured  into  this  funnel, 
which  has  been  carefully  washed,  and  by  pressing  the 
pinchcock  with  which  the  funnel  is  provided  the  desired 
amount  of  material  (5-10  c.c.)  may  be  allowed  to  flow 
into  the  tubes  held  under  its  opening.  It  is  not  neces- 
sary to  sterilize  the  funnel,  for  the  medium  is  to  be  sub- 
jected to  this  process  as  soon  as  it  is  in  the  test-tubes. 

FIG.  22. 


Funnel  for  filling  tubes  with  culture-media. 

Care  should  be  taken  that  none  of  the  medium  is 
dropped  upon  the  mouth  of  the  test-tube,  otherwise  the 
cotton  plug  becomes  adherent  to  it,  and  is  not  only 
difficult  to  remove,  but  presents  a  very  untidy  appear- 
ance and  interferes  materially  with  the  manipulations. 


FILLING  THE  TUBES.  123 

As  soon  as  the  tubes  have  been  filled  they  are  to  be 
sterilized  in  the  steam  sterilizer  for  fifteen  minutes  on 
each  of  three  successive  days.  During  the  intervening 
days  they  may  be  kept  at  the  ordinary  room-tempera- 
ture. 

When  sterilization  is  complete  and  the  medium  in 
the  tubes  is  still  liquid,  some  of  them  may  be  placed  in 
a  slanting  position,  at  an  angle  of  about  ten  degrees 
with  the  surface  on  which  they  rest,  and  the  medium 
allowed  to  solidify  in  this  position.  These  are  for  the 
so-called  slant-cultures.  The  remainder  may  solidify  in 
the  erect  position ;  these  serve  for  making  plates. 

For  Esmarch  tubes  not  more  than  5  c.c.  of  material 
should  be  placed  in  each  tube,  as  more  than  this  renders 
it  difficult  to  distribute  the  gelatin  evenly  over  the  inner 
surface  of  the  tubes  when  they  are  rolled. 


CHAPTER  VII. 

Technique  of  making  plates — Esmarch  tubes,  Petri  plates,  etc. 

PLATES. — The  plate  method  can  be  employed  with 
both  agar-agar  and  gelatin.  It  cannot  be  practised  with 
blood-serum,  because  the  serum  when  once  solidified 
cannot  be  again  liquefied. 

Plates  are  usually  referred  to  as  "  a  set."  This 
term  implies  three  individual  plates,  each  representing 
a  mixture  of  organisms  in  a  higher  state  of  dilution. 
The  first  plate  is  known  usually  as  "  the  original,"  or 
"  plate  No.  1,"  the  first  dilution  from  this  as  "  plate 
No.  2,"  and  the  second  as  "  plate  No.  3." 

In  the  preparation  of  a  set  of  plates  the  following 
are  the  steps  to  be  observed  : 

Three  tubes,  each  containing  from  7  to  9  c.c.  of  gela- 
tin or  agar-agar,  are  placed  in  a  warm  water-bath 
until  the  medium  has  become  liquid.  If  agar-agar  is 
employed,  this  is  accomplished  at  the  boiling-point  of 
water;  if  gelatin  is  used,  a  much  lower  temperature 
suffices  (35°-40°  C.).  When  liquefaction  is  complete 
the  temperature  of  the  water,  in  the  case  of  agar-agar, 
must  be  reduced  to  41°-42°  C.,  at  which  temperature 
the  agar-agar  remains  liquid,  and  the  organisms  may 
be  introduced  into  it  without  fear  of  destroying  their 
vitality.  The  medium  being  now  liquid  and  of  the 
proper  temperature,  a  very  small  portion  of  the  mixture 
of  organisms  to  be  studied  is  taken  up  with  a  sterilized 

124 


TECHNIQUE  OF  MAKING  PLATES.  125 

platinum  wire  (Fig.  23,  a)  about  5  cm.  long,  twisted 
into  a  small  loop  at  one  end  and  fused  into  a  bit 
of  glass  rod,  which  serves  as  a  handle,  at  the  other 
extremity.  This  loop  is  one  of  the  most  useful  of  bac- 
teriological instruments,  as  there  is  hardly  a  manipula- 
tion into  which  it  does  not  enter.  Under  no  circum- 
stances is  it  to  be  employed  without  having  been 
passed  through  a  gas-flame  until  quite  hot,  for  the 
purpose  of  sterilization.  One  should  form  a  habit 


FIG.  23. 
a 


b 
Looped  and  straight  platinum  wires  in  glass  handles. 

of  never  taking  up  one  of  these  platinum-wire  needles, 
as  they  are  called,  for  they  are  curved  and  straight  (Fig. 
23,  6)  as  well  as  looped,  without  passing  it  through 
a  flame ;  and  the  sooner  the  beginner  learns  to  do  this 
as  a  reflex  action,  the  sooner  does  he  eliminate  one 
of  the  possible  sources  of  error  in  his  work.  It  must 
be  remembered,  though,  that  it  should  not  be  used  when 
hot,  otherwise  the  organisms  taken  upon  it  will  be  killed 
by  the  high  temperature ;  after  sterilization  in  the  flame 
one  waits  for  a  few  seconds  until  it  is  cool  before  using. 
A  minute  portion  of  the  material  under  consideration 
is  transferred  with  the  sterilized  loop  into  tube  No.  1, 
"  the  original,"  where  it  is  thoroughly  disintegrated  by 
gently  rubbing  it  against  the  sides  of  the  tube.  The 
more  carefully  this  is  done  the  more  uniform  will  be 


126  BACTERIOLOGY. 

the  distribution  of  the  organisms  and  the  better  the 
results.  The  loop  is  then  again  sterilized  and  three 
of  its  loopfuls  are  passed,  without  touching  the  sides  of 
the  tube,  from  "  the  original "  into  tube  No.  2,  where 
they  are  carefully  mixed.  Again  the  loop  is  sterilized, 
and  again  three  dips  are  made  from  tube  No.  2  into 
tube  No.  3.  This  completes  the  dilution.  The  loop  is 
now  sterilized  before  laying  it  aside. 

FIG.  24. 


Levelling-tripod  with  glass  chamber  for  plates. 

During  this  manipulation,  which  must  be  done 
quickly  if  agar-agar  be  employed,  the  temperature  of 
the  water  in  the  bath  in  which  the  tubes  stand  should 
never  be  lower  than  39°  C.,  and  never  higher  than 
43°  C.  If  it  falls  below  38°  C.,  the  agar-agar  solid- 
ifies, and  can  only  be  redissolved  at  a  temperature 
that  would  be  destructive  to  the  organisms  which  may 
have  been  introduced  into  the  tubes.  This  is  not  of 
so  much  moment  with  gelatin,  since  it  may  readily  be 


TECHNIQUE  OF  MAKING  PLATES.  127 

ivdissnlved  at  a  temperature  not  detrimental  to  the 
organisms  with  which  the  tubes  may  have  been  inocu- 
lated. 

THE  COOLING-STAGE  AND  LEVELLING-TRIPOD. — 
While  the  medium  of  which  the  plates  are  to  be  made 
is  melting  it  is  well  to  arrange  the  cooling-stage  (Fig. 
24)  upon  which  the  gelatin  or  agar-agar  is  to  be  subse- 
quently solidified. 

This  stage  consists  of  a  glass  dish  filled  with  ice- 
water  and  covered  with  a  ground-glass  plate,  which  in 
turn  has  a  dome-shaped  cover.  The  dish  rests  upon  a 
tripod  which  can  be  brought  to  an  exact  level,  as  indi- 
cated by  the  spirit-level,  by  raising  or  lowering  its  legs 
by  means  of  thumb-screws,  with  which  they  are  pro- 
vided. Three  stages  are  usually  employed.  When 
ready  for  use  they  should  be  exactly  level. 

THE  GLASS  PLATES. — On  each  of  the  stages  is  to  be 
placed  a  glass  plate  upon  which  the  liquefied  gelatin  or 

FIG.  25. 


Russia  iron  box  for  holding  plates,  etc.,  during  sterilization  in  dry  heat. 

agar-agar  is  to  be  poured  and  allowed  to  solidify.  It 
is,  therefore,  necessary  that  the  plates  should  not  only 
be  sterile  when  placed  upon  the  stages,  but  that  during 
manipulation  they  should  be  carefully  protected  by  a 
cover  against  dust  and  bacteria  from  without. 


128  BACTERIOLOGY. 

A  number  of  plates  at  a  time  are  usually  sterilized  in 
the  dry  sterilizer  at  a  temperature  of  150°  to  180°  C. 
for  one  hour.  During  sterilization  and  until  used 
they  are  retained  in  an  iron  box  (Fig.  25),  which  is 
especially  designed  for  the  purpose.  They  should  never 
be  placed  upon  the  stage  until  cold ;  otherwise  they 
crack. 

When  the  plates  which  have  been  placed  upon  the 
stages  are  quite  cold  the  melted  gelatin  or  agar-agar  in 
the  tubes  which  represent  the  three  dilutions  should  be 
poured  upon  them,  each  tube  being  emptied  upon  a 
separate  plate.  If  the  medium  is  quite  fluid,  it  spreads 
over  the  surface  of  the  plates  in  a  thin,  even  layer. 
Sometimes  it  may  be  more  evenly  spread  as  it  flows 
from  the  tube  by  the  aid  of  a  sterilized  glass  rod. 


FIG.  26. 


Glass  benches  for  supporting  plates. 

As  the  content  of  each  tube  is  emptied  the  cover  of 
the  cooling-stage  is  quickly  replaced  and  the  plate  al- 
lowed to  stand  until  the  gelatin  or  agar-agar  is  quite 
solid.  This  takes  longer  with  gelatin  than  with  agar. 
When  the  medium  is  quite  solid  these  plates  are  placed 
upon  glass  benches  (Fig.  26),  each  labelled  with  the  num- 
ber of  the  plate  in  the  series  of  dilutions.  The  benches, 
with  the  plates  upon  them,  are  then  piled  one  above  the 
other  in  a  "  culture-dish,"  in  which  the  plates  are  to  be 
kept  during  the  growth  of  the  bacteria.  The  benches 


CULTURE-DISH.  129 

are  sterilized  before  using,  in  the  same  manner  as 
plates. 

CULTURE-DISH. — This  dish,  which  is  about  22  cm. 
in  diameter  and  has  vertical  sides  about  6  cm.  in  height, 
is  provided  with  a  cover  of  exactly  the  same  shape, 
but  of  a  little  larger  diameter.  This  cover,  when 
placed  upon  the  dish  containing  the  plates,  prevents 
the  access  of  dust.  Prior  to  using,  the  dish  and  cover 
should  have  been  disinfected  for  one-half  hour  with 
1  : 1000  sublimate,  and  all  the  solution  allowed  to  drain 
from  them. 

A  disk  of  sterilized  filter-paper  moistened  with  ster- 
ilized water  is  sometimes  placed  in  the  bottom  of  the 
culture-dish  to  prevent  drying  of  the  medium.  This 
is  not  often  necessary,  however,  as  the  plates  can 
usually  be  dispensed  with  before  evaporation  has  pro- 
gressed very  far. 

If  agar-agar  be  employed,  the  dish  and  its  contents 
may  be  kept  at  a  temperature  of  37°-38°  C. ;  if  gel- 
atin, the  temperature  at  which  the  plates  are  to  be 
maintained  should  not  be  over  22°  C.,  otherwise  the 
gelatin  liquefies  and  the  plates  are  rendered  useless. 

When  development  has  occurred  the  object  of  the 
dilution  will  be  easily  seen,  and  the  various  species  of 
bacteria  in  the  mixture  will  be  recognized  by  differences 
in  the  character  of  the  colonies  growing  from  them. 

This,  in  short,  is  the  plate  method  of  Koch  for  the 
separation  of  the  individual  species  contained  in  a 
mixture  of  bacteria.  Many  modifications  of  this  method 
exist ;  all,  however,  are  based  upon  the  same  prin- 
ciples. The  modifications  have  for  their  object  the 
accomplishment  of  the  same  end,  but  with  a  smaller 
armamentarium  of  apparatus  ;  according  to  individual 


130  BACTERIOLOGY. 

preference,  the  one  or  the  other  of  these  modifications 
has  supplanted  the  original  plate  method  as  practised 
and  recommended  by  Koch. 

PETRI'S  MODIFICATION  OF  THE  PLATE  METHOD. — 
The  modification  which  approaches  nearest  to  the  orig- 
inal method,  and  at  the  same  time  lessens  very  mate- 
rially the  number  of  steps  in  the  process,  is  that  sug- 
gested by  Petri.  It  consists  in  substituting  for  the 
plates  small,  round,  double  glass  dishes,  having  about 
the  same  surface-area  as  the  plates  (Fig.  27).  The 
liquid  medium  is  poured  directly  into  these  little  dishes 
and  their  covers  replaced  ;  they  are  then  set  aside  for  ob- 
servation. In  all  other  respects  the  process  is  the  same 
as  Koch's  original  method.  Petri's  dishes  are  about  8 

FIG.  27. 


Petri  double  dish,  now  generally  used  instead  of  plates. 

cm.  in  diameter  and  about  1.5  to  2  cm.  in  height,  the 
sides  being  vertical.  They  may  readily  be  sterilized 
by  either  hot-air  or  steam.  They  are  very  useful  for  this 
work,  as  they  do  away  with  the  necessity  for  the  cool- 
ing-stage and  levelling-tripod,  though  in  warm  weather 
the  cooling-stage  may  be  used  to  hasten  the  solidifica- 
tion of  gelatin.  A  cooling-stage  of  very  convenient 
design  for  use  with  these  dishes  consists  of  a  closed,  flat 
metal  box,  either  of  copper  or  block  tin,  and  round 
or  square  in  shape,  so  arranged  that  it  can  be  filled 
with  cold  water,  or  that  cold  water  can  constantly  be 


ESMARCH  TUBES.  131 

passed  through  it  by  means  of  a  rubber  tube  connected 
with  a  spigot.  The  inlet  for  the  water  should  be  just 
above  the  bottom  of  the  box,  and  the  outlet  just  beneath 
the  top  and  slightly  turned  upward  and  then  downward, 
so  as  to  insure  filling  the  space  with  water.  The  box 
should  be  sufficiently  strong  to  resist  the  pressure  of 
the  water.  A  convenient  size  is  from  20  to  25  cm. 
in  diameter,  and  about  1.5  to  2  cm.  high.  It  is  simple 
in  construction,  and  can  be  made  by  any  copper-spinner. 
An  idea  of  its  construction  is  given  in  Fig.  28. 

FIG.  28. 


Metal  cooling-stage. 

When  gelatin  or  agar-agar  is  to  be  cooled  it  is  only 
necessary  to  place  the  dishes  containing  it  on  top  of  this 
box  and  keep  cold  water  circulating  through  it. 

ESMARCH  TUBES. — The  modification  of  Koch's 
method  which  insures  the  greatest  security  from  con- 
tamination by  extraneous  organisms  and  requires  the 
least  amount  of  apparatus  is  that  suggested  by  v. 
Esmarch.  It  differs  from  the  other  methods  thus  :  the 
dilutions  having  been  prepared  in  tubes  containing  a 
smaller  amount  of  medium  than  usual — as  a  rule,  not 
more  than  5  to  6  c.c. — are,  instead  of  being  poured  upon 
plates  or  into  dishes,  spread  over  the  inner  surface  of 


132  BACTERIOLOGY. 

the  tubes  containing  them,  and,  without  removing  the 
cotton  plugs,  solidified  in  this  position.  The  tubes  then 
present  a  thin  cylindrical  lining  of  gelatin  or  agar-agar, 
upon  which  the  colonies  develop.  In  all  other  respects 
the  conditions  for  the  growth  of  the  organisms  are  the 
same  as  in  flat  plates. 

Esmarch  directs  that  after  completion  of  the  dilu- 
tions the  tops  of  the  cotton  plugs  in  the  test-tubes 
should  be  cut  off  flush  with  the  mouths  of  the  tubes  and 
sterilized  rubber  caps  be  placed  over  them.  They  are 
then  to  be  held  in  a  horizontal  position  and  twisted 
between  the  fingers  upon  their  long  axis  under  ice- 
water.  The  gelatin  becomes  solidified  thereby  and 
adheres  to  the  sides  of  the  tube.  When  the  gelatin  is 
quite  hard  the  tubes  are  removed  from  the  water,  wiped 
dry,  the  rubber  caps  removed,  and  the  tubes  set  aside 
for  observation. 

For  some  time  past  we  have  deviated  from  the  direc- 
tion given  by  v.  Esmarch  for  this  part  of  his  method, 
and  instead  of  rolling  the  tubes  under  ice-water,  we  roll 
them  upon  a  block  of  ice  (Fig.  29),  after  the  method 
devised  by  Booker  in  1887  in  the  Pathological  Labora- 
tory of  the  Johns  Hopkins  University.  In  this  method 
a  small  block  of  ice  only  is  needed.  It  is  levelled 
and  held  in  position  by  being  placed  upon  a  towel  in 
a  dish.  A  horizontal  groove  is  melted  in  the  upper 
surface  of  the  ice  with  a  test-tube  of  hot  water.  The 
tubes  to  be  rolled  are  then  held  in  an  almost — not  quite 
— horizontal  position  and  twisted  between  the  fingers 
until  the  sides  are  moistened  by  the  contents  to  within 
about  1  cm.  of  the  cotton  plug,  care  being  taken  that 
the  gelatin  does  not  touch  the  cotton  ;  otherwise  the  latter 
becomes  adherent  to  the  sides  of  the  tube  and  is  difficult 


ESMARCH  TUBES.  133 

to  remove.  The  tube  is  then  placed  in  the  groove  in 
the  ice  and  rolled,  neither  rubber  cap  nor  cutting  off  of 
the  cotton  plug  being  necessary. 


FIG.  29. 


Demonstrating  Booker's  method  of  rolling  Esmarch  tubes  on  a  block  of  ice. 

The  advantages  of  this  process  over  that  followed  by 
v.  Esmarch  are  that  it  requires  less  time,  is  cleaner, 
no  rubber  caps  are  needed,  the  rolled  tubes  are  more 
uniform,  and  the  gelatin  does  not  touch  the  cotton  plug, 
as  is  always  the  case  in  tubes  rolled  under  water,  be- 
cause of  the  impossibility  of  keeping  them  at  one  level. 

There  is  an  erroneous  impression  that  Esmarch  tubes 
are  not  a  success  when  made  from  ordinary  nutrient 
agar-agar  because  of  the  tendency  of  this  medium  to 
shrink  and  fall  to  the  bottom  of  the  tube.  This  slip- 
ping down  of  the  agar-agar  is  due  to  the  water  squeezed 
from  it  during  solidification  getting  between  the  medium 
and  the  walls  of  the  tube.  This  can  easily  be  over- 
come by  allowing  the  rolled  tubes  to  remain  in  a  nearly 
horizontal  position  for  twenty-four  hours  after  rolling 
them,  the  mouth  of  the  tube  being  about  1  cm.  higher 


134  BACTERIOLOGY. 

than  the  bottom.  During  this  time  the  margin  of  the 
agar-agar  nearest  the  cotton  plug  dries  and  becomes 
adherent  to  the  walls  of  the  tube,  while  the  water  col- 
lects at  the  most  dependent  point — i.  e.,  the  bottom  of 
the  tubes.  After  this  they  may  be  retained  in  the  up- 
right position  without  danger  of  the  agar-agar  slipping 
down.  We  have  employed  this  method  for  several  years 
with  entire  satisfaction.  In  all  these  manipulations, 
if  the  dilutions  of  the  number  of  organisms  have  been 
properly  conducted,  the  results  will  be  the  same.  The 
original  plate  or  tube,  as  a  rule,  will  be  of  no  use  be- 
cause of  the  great  number  of  colonies  in  it ;  plate  or 
tube  No.  2  may  be  of  service ;  but  plate  or  tube, No.  3 
will  usually  contain  the  organisms  in  such  small  num- 
bers that  there  will  be  nothing  to  prevent  the  charac- 
teristic development  of  the  colonies  originating  from 
them. 

For  reasons  of  economy  the  "  original,"  tube  No.  1,  is 
sometimes  substituted  by  a  tube  containing  normal  salt- 
solution  (0.6  to  0.7  per  cent,  of  sodium  chloride  in 
water),  which  is  thrown  aside  as  soon  as  the  dilutions 
are  completed,  and  only  plates  or  tubes  Nos.  2  and  3 
are  made. 

Another  method  for  the  separation  of  bacteria  and 
their  isolation  as  single  colonies  consists  in  the  making 
of  dilutions  upon  the  surface  of  solid  media,  such  as 
potato,  coagulated  blood-serum,  agar-agar,  and  gelatin. 
In  pursuance  of  this  method  one  selects  a  number  of 
tubes  containing  the  medium  set  in  a  slanting  position. 
With  a  platinum  needle  a  bit  of  the  substance  to  be 
studied  is  smeared  upon  tube  No.  1 ;  without  sterilizing 
the  needle  it  is  passed  in  succession  over  the  surface 
of  the  medium  in  tubes  Nos.  2,  3,  4,  etc.  When  de- 


ESMAECH  TUBES.  135 

velopment  has  occurred  essentially  the  same  conditions 
as  regards  separation  of  the  colonies  will  be  found  as 
when  plates  are  poured.  If  a  slanted  medium  be  em- 
ployed, about  the  most  dependent  angle  of  which  water 
of  condensation  has  accumulated,  as  blood-serum,  agar- 
agar,  and  potato,  the  dilutions  may  be  made  in  this 
fluid,  and  this  is  then  to  be  carefully  smeared  over  the 
solid  surface  of  the  medium.  The  tubes  thus  treated 
should  be  kept  in  an  upright  position  to  prevent  the 
fluid  flowing  over  the  surface.  When  sufficiently  de- 
veloped, single  colonies  may  be  isolated  with  compara- 
tive ease  from  tubes  prepared  in  this  manner.  (See  also 
method  for  the  isolation  of  bacillus  diphtherice  on  blood- 
serum.) 


CHAPTER    VIII. 

The  incubating-oven — Gas-pressure  regulator — Thermo-regulator — The 
safety  burner  employed  in  heating  the  incubator. 

THE  INCUBATOR. — When  the  plates  have  been  made 
it  must  be  borne  in  mind  that  for  the  development  of 
certain  forms  of  bacteria  a  higher  temperature  is  neces- 
sary than  for  the  growth  of  others.  The  pathogenic 
or  disease-producing  organisms  grow  more  luxuriantly 
at  the  temperature  of  the  human  body  (37.5°  C.)  than 
at  lower  temperatures  ;  whereas  for  the  ordinary  sap- 
rophytic  forms  almost  any  temperature  between  18° 
and  37°  C.  is  suitable.  It  therefore  becomes  neces- 
sary to  provide  a  place  in  which  a  constant  tem- 
perature favorable  to  the  growth  of  the  pathogenic 
organisms  can  be  maintained.  For  this  purpose  a  num- 
ber of  different  forms  of  apparatus  have  been  devised. 
They  are  all  based  upon  the  same  principles,  however, 
and  a  general  description  of  the  essential  points  involved 
in  their  construction  will  be  all  that  is  needed  here. 

The  apparatus  known  as  thermostat,  incubator,  or 
brooding-oven,  is  a  copper  chamber  (Fig.  30)  with 
double  walls,  the  space  betAveen  which  is  filled  with 
water.  The  inctibating-chamber  has  a  closely  fitting 
double  door,  inside  of  which  is  usually  a  door  of  glass 
through  which  the  contents  of  the  chamber  may  be  in- 
spected without  actually  opening  it.  The  whole  appa- 
ratus is  encased  in  either  asbestos-boards  or  thick  felt, 

136 


THE  INCUBATOR. 


137 


to  prevent  radiation  of  heat  and  consequent  fluctuations 
in  temperature.  In  the  top  of  the  chamber  is  a  small 
opening  through  which  a  thermometer  projects  into  its 
interior.  At  either  corner,  leading  into  the  space  con- 
taining the  water,  are  other  openings  for  the  reception 

FIG.  30. 


Incubator  used  in  bacteriological  work. 

of  another  thermometer  and  a  thermo-regulator,  and  for 
refilling  the  apparatus  as  the  water  evaporates.  On  the 
side  is  a  water-gauge  for  showing  the  level  of  the  water 


138 


BACTERIOLOGY. 


between  the  walls.  The  object  of  the  water-chamber, 
which  is  formed  by  the  double-wall  arrangement,  is  to 
insure,  by  means  of  the  warmed  water,  an  equable  tem- 
perature in  all  parts  of  the  apparatus — at  the  top  as  well 
as  at  the  sides,  back,  and  bottom  ;  the  apparatus  should 
be  kept  filled  with  water,  otherwise  the  purpose  for 
which  it  is  constructed  will  not  be  accomplished.  When 
the  chamber  between  the  walls  is  filled  with  water  heat 
is  supplied  by  a  gas-flame  placed  beneath  it. 

FIG.  31. 


Koch's  safety  burner. 


The  burner  employed  in  heating  the  incubator  was 
originally  devised  by  Koch,  and  is  known  as  "  Koch's 
safety  burner "  (Fig.  31).  It  is  a  Bunsen  burner  pro- 
vided with  an  arrangement  for  automatically  turning 
off  the  gas-supply  and  thus  preventing  accidents  should 


THERMO-REG  ULA  TORS.  139 

the  flame  become  extinguished  at  a  time  when  no  one 
is  near.  The  gas-cock  by  which  the  gas  is  turned  on 
and  off  is  provided  with  a  long  arm  which  is  weighted, 
and  which,  when  the  gas  is  turned  on  and  burning,  rests 
upon  an  arm  attached  to  the  side  of  a  revolving,  hori- 
zontal disk  that  is  connected  with  the  free  ends  of  two 
metal  spirals  which  are  fixed  by  their  other  ends  in  oppo- 
site directions  on  either  side  of  the  flame  and  heated  by 
it.  If  by  draughts  or  any  other  accident  the  flame  be- 
comes extinguished,  the  metal  spirals  cool,  and  in  cool- 
ing contract,  twist  the  horizontal  disk  in  the  opposite 
direction,  and  allow  the  weighted  arm  of  the  gas-cock 
to  fall.  By  its  falling  the  gas-supply  is  turned  off. 

THERJVIO-REGULATORS. — The  regulation  and  main- 
tenance of  the  proper  temperature  within  the  incubator 
are  accomplished  by  the  employment  of  an  automatic 
thermo-regulator. 

The  common  form  of  thermo-regulator  used  for  this 
purpose  is  constructed  upon  principles  involving  the 
expansion  and  contraction  of  fluids  under  the  influence 
of  heat  and  cold.  By  means  of  this  expansion  and  con- 
traction the  amount  of  gas  passing  from  the  source  of 
supply  to  the  burner  may  be  either  diminished  or  in- 
creased as  the  temperature  of  the  substance  in  which 
the  regulator  is  placed  either  rises  or  falls. 

The  simplest  form  of  thermo-regulator  which  serves 
to  illustrate  the  principles  involved  is  seen  in  Fig.  32. 
It  consists  of  a  glass  cylinder,  e,  having  a  communi- 
cating branch  tube  6,  and  rubber  stopper  /,  through 
which  projects  the  bent  tube  a.  The  tube  a  is  ground 
to  a  slanting  point  at  the  extremity  which  projects  into 
the  tube  e,  and  is  provided  a  short  distance  above  this 
point  with  a  capillary  opening,  g,  in  one  of  its  sides. 


140 


BACTERIOLOGY. 


When  ready  for  use  the  cylinder  e  is  filled  with  mer- 
cury up  to  about  the  level  shown  in  the  figure.  It  is 
then  allowed  to  stand,  or  is  suspended,  in  the  bath  the 
temperature  of  which  it  is  to  regulate.  The  rubber 
tubing  coming  from  the  gas-supply  is  attached  to  the 

FIG.  32. 


Mercurial  thermo-regulator. 


outer  end  of  the  glass  tube  a,  and  the  tube  going  to  the 
burner  is  slipped  over  the  branch  tube  b.  The  gas  is 
turned  on  and  the  burner  lighted  and  placed  under  the 
bath.  The  gas  now  streams  through  the  tube  a  into  the 
cylinder  e  and  out  at  b  to  the  burner ;  but  as  the  tern- 


THERMO-REG  ULA  TORS.  141 

perature  of  the  bath  rises  the  mercury  contained  in 
the  cylinder  e,  under  the  influence  of  the  elevation  of 
temperature,  begins  to  expand,  and,  as  a  continuous  rise 
in  temperature  proceeds,  the  expansion  of  the  mercury 
accompanies  it  and  gradually  closes  the  slanting  opening 
h  of  tube  a.  In  this  way  the  supply  of  gas  becomes 
diminished  and  the  rise  in  temperature  of  the  bath  will 
be  less  rapid,  until  finally  the  opening  at  h  will  be  closed 
entirely,  when  the  supply  of  gas  to  the  burner  will  now 
be  limited  to  that  passing  through  the  capillary  open- 
ing g.  This  is  not  sufficient  to  maintain  the  highest 
temperature  reached,  and  as  cooling  begins  a  gradual 
contraction  of  the  mercury  occurs  until  there  is  again 
an  outflow  of  gas  from  the  opening  A,  when  the  tem- 
perature again  rises.  This  contraction  and  expansion  of 
the  mercury  in  the  regulator  continues  until  eventually 
a  point  is  reached  at  which  its  position  in  the  cylinder 
e  allows  of  the  passage  of  just  enough  gas  from  the 
opening  h  to  maintain  a  constant  temperature  and, 
therefore,  a  constant  degree  of  expansion  of  the  mercury 
in  the  tube  e.  This,  in  short,  is  the  principle  on  which 
ther mo-regulators  are  constructed  ;  but  it  must  be  borne 
in  mind  that  a  great  deal  of  detail  exists  in  the  construc- 
tion of  an  accurate  instrument.  The  number  of  differ- 
ent forms  of  this  apparatus  is  comparatively  large,  and 
each  form  has  its  special  merits. 

The  value — that  is,  the  delicacy — of  the  thermo-reg- 
ulator  depends  upon  a  number  of  factors,  all  of  which  it 
would  be  useless  to  describe  in  a  book  of  this  kind  ; 
but  in  general  it  may  be  said  that  the  essential  points  to 
be  observed  in  selecting  a  thermo-regulator  depend  in  the 
main  upon  the  temperatures  at  which  it  is  to  be  used. 
For  low  temperatures,  regulators  containing  such  fluids 


142 


BACTERIOLOGY. 


as  ether,  alcohol,  and  calcium  chloride  solution,  which 
expand  and  contract  rapidly  and  regularly  under  slight 
variations  in  temperature,  are  commonly  employed ; 
whereas  for  temperatures  approaching  the  boiling-point 
of  water  mercury  is  most  frequently  used. 

The  temperature  of  the  incubator  is  to  be  regulated, 
then,  by  the  use  of  some  such  form  of  apparatus  as  that 
just  described.  It  should  be  of  sufficient  delicacy  to 
prevent  a  fluctuation  of  more  than  0.2°  C.  in  the  tem- 
perature of  the  air  within  the  chamber  of  the  apparatus. 

GAS-PRESSURE  KEGULATORS. — A  gas-pressure  reg- 
ulator is  not  rarely  intervened  between  the  gas-supply 

FIG.  33. 


Moitessier's  gas-pressure  regulator. 


and  the  thermo-regulator.  This  apparatus  has  for  its 
object  the  maintenance  of  a  constant  pressure  of  the 
gas  going  to  the  thermo-regulator.  There  are  several 


GAS-PRESSUEE  REGULATORS.  143 

forms  of  regulator  in  use,  but  they  do  not  accomplish 
the  object  for  which  they  are  designed. 

The  instrument  most  commonly  employed,  the  appa- 
ratus of  Moitessier  (Fig.  33),  is  based  on  somewhat  the 
same  principles  as  the  large  regulators  seen  at  the  manu- 
factories of  illuminating-gas.  Such  apparatus  act  very 
well  when  employed  on  the  large  scale,  as  one  sees  them 
at  the  gas-works ;  but  when  applied  to  the  limited  and 
sudden  fluctuations  seen  in  the  gas  coming  from  an 
ordinary  gas-cock  are  practically  useless.  They  are  too 
gross  in  their  construction,  and  act  only  under  compar- 
atively great  and  gradual  fluctuations  in  pressure.  If 
a  good  form  of  thermo-regulator  be  employed,  there,  is 
no  necessity  for  the  use  of  any  of  the  pressure-regulators 
thus  far  introduced. 


CHAPTER  IX. 

The  study  of  colonies — Their  naked-eye  peculiarities  and  their  appear- 
ance under  different  conditions — Differences  in  the  structure  of 
colonies  from  different  species  of  bacteria — Stab-cultures — Slant- 
cultures. 

THE  plates  of  agar-agar  which  have  been  prepared 
from  a  mixture  of  organisms  and  have  been  placed  in 
the  incubator,  and  those  of  gelatin  which  have  been 
maintained  at  the  ordinary  temperature  of  the  room, 
are  usually  ready  for  examination  after  from  twenty-four 
to  forty-eight  hours.  They  will  be  found  marked  here 
and  there  by  small  points  or  little  islands  of  more  or 
less  opaque  appearance.  In  some  instances  these  will 
be  so  transparent  that  it  is  with  difficulty  one  can  see 
them  with  the  naked  eye.  Again,  they  may  be  of  a 
dense,  opaque  appearance ;  at  one  time  sharply  circum- 
scribed and  round,  again  irregular  in  their  outline  ;  here 
a  point  will  present  one  color,  there  perhaps  another. 
On  gelatin  some  of  the  points  will  be  seen  to  be  lying 
on  the  surface  of  the  medium,  others  will  have  sunk 
into  little  depressions,  while  at  still  other  points  the 
clear  gelatin  will  be  marked  by  more  or  less  saucer- 
shaped  pits  containing  opaque  fluid. 

Place  the  plate  containing  these  points  upon  the 
stage  of  a  microscope  and  examine  them  with  a  low- 
power  objective,  and  again  differences  will  be  observed. 
Some  of  these  minute  points  will  be  finely  granular, 
others  coarsely  so  ;  some  will  present  a  radiated  appear- 
ance, while  a  neighbor  may  be  concentrically  arranged  ; 

144 


THE  STUDY  OF  COLONIES.  145 

here  nothing  particularly  characteristic  will  present, 
there  the  point  may  resolve  itself  into  a  mass  having 
somewhat  the  appearance  of  a  little  pellicle  of  raw  cot- 
ton. All  these  differences,  and  many  more,  aid  us  in 
saying  that  these  objects  must  be  different  in  their 
nature.  With  a  pointed  platinum  needle  take  up  a 
bit  of  one  of  these  small  islands,  prepare  it  for  micro- 
scopic examination  (see  chapter  on  Stained  Cover-slip 
Preparations),  and  examine  it  under  the  high-power  oil- 
immersion  objective,  with  access  of  the  greatest  amount 
of  light  afforded  by  the  illuminator  of  the  microscope. 
The  preparation  will  be  seen  to  be  made  up  entirely  of 
bodies  of  the  same  shape ;  they  will  all  be  spheres,  or 
ovals,  or  rods,  but  not  a  mixture  of  these  forms,  if  proper 
care  in  the  manipulation  has  been  taken.  Examine  in 
the  same  way  a  neighboring  spot  which  possesses  dif- 
ferent naked-eye  appearances,  and  it  will  often  be  found 
to  consist  of  bodies  of  an  entirely  different  appearance 
from  those  seen  in  the  first  preparation. 

These  spots  or  islands  on  the  surface  of  the  plates  are 
colonies  of  bacteria,  differing  severally,  not  only  in  their 
gross  appearances,  the  one  from  the  other,  but,  as  our 
cover-slip  preparations  show,  in  the  morphological  char- 
acteristics of  the  individual  organisms  composing  them. 

If  from  one  of  these  colonies  a  second  set  of  plates  be 
prepared,  the  peculiarities  which  were  first  observed  in 
it  will  be  reproduced  in  all  of  the  new  colonies  which 
develop ;  each  will  be  found  to  consist  of  the  same 
organisms  as  the  colony  from  which  the  plates  were 
made.  In  other  words,  these  peculiarities  are  constant 
under  like  conditions. 

The  appearance  of  the  colonies  developing  from 
all  organisms  is  regulated  by  their  location  in  the 
10 


146  BACTERIOLOGY. 

medium  in  which  they  are  growing.  When  deep 
down  in  the  medium  they  are  usually  round,  oval,  or 
lozenge-shaped ;  whereas  when  on  the  surface  of  the 
gelatin  or  agar  they  may  take  quite  a  different  form. 
This  is  purely  a  mechanical  effect  due  to  the  pressure 
of,  or  resistance  offered  by,  the  medium  surrounding 
them,  and  is  always  to  be  borne  in  mind,  otherwise 
errors  are  apt  to  arise. 

PURE  CULTURES. — If  from  one  of  these  small  col- 
onies a  bit  be  taken  upon  the  point  of  a  sterilized  plati- 
num needle  and  introduced  into  a  tube  of  sterilized 
gelatin  or  agar-agar,  the  growth  that  results  will  be 
what  is  known  as  a  "  pure  culture,"  the  condition  to 
which  all  organisms  must  be  brought  before  a  system- 
atic study  of  their  many  peculiarities  is  begun.  Some- 
times several  series  of  plates  are  necessary  before  the 
organisms  can  be  obtained  pure,  but  by  patiently  follow- 
ing this  plan  the  results  will  ultimately  be  satisfactory. 

TEST-TUBE  CULTURES  ;  STAB-CULTURES  ;  SMEAR- 
CULTURES. — After  separating  the  organisms  the  one 
from  the  other  by  the  plate  method  just  described,  they 
must  be  isolated  from  the  plates  as  pure  stab-  or  smear- 
cultures. 

This  is  done  in  the  following  way  :  decide  upon  the 
colony  from  which  the  pure  culture  is  to  be  made. 
Select  preferably  a  small  colony  and  one  as  widely  sep- 
arated from  other  colonies  as  possible.  Sterilize  in  a 
gas-flame  a  straight  platinum-wire  needle.  The  glass 
handle  of  the  needle  should  be  drawn  through  the  flame 
as  well  as  the  needle  itself,  otherwise  contamination  from 
this  source  may  occur.  When  it  is  cool,  which  is  in  five 
or  ten  seconds,  take  up  carefully  a  portion  of  the  colony. 
Guard  against  touching  anything  but  the  colony.  If 


TEST-TUBE,  STAB-  AND  SMEAR-CULTURES.    147 

during  manipulation  the  needle  touches  anything  else 
ivhatei'cr  than  the  colony  from  which  the  culture  is  to 
be  made,  it  must  be  sterilized  again.  This  holds  not 
only  for  the  time  before  touching  the  colony,  but  also 
during  its  passage  into  the  test-tube  from  the  colony ; 
otherwise  there  is  no  guarantee  that  the  growth  result- 
ing from  the  inoculation  of  this  bit  of  colony  into  a 
fresh  sterile  medium  will  be  pure. 

In  the  meantime  have  in  the  other  hand  a  test-tube 
of  sterile  medium  :  gelatin,  agar-agar,  or  potato.  This 
tube  is  held  across  the  palm  of  the  hand  in  an  almost 
horizontal  position  with  its  mouth  pointing  out  between 
the  thumb  and  index-finger  and  its  contents  toward  the 
body  of  the  worker.  With  the  disengaged  fingers  of  the 
other  hand  holding  the  needle  the  cotton  plug  is  removed 
from  the  tube  by  a  twisting  motion  and  placed  between 
the  index  and  second  fingers  of  the  hand  holding  the 
tube,  in  such  a  way  that  the  portion  of  the  plug  which 
fits  into  the  mouth  of  the  test-tube  looks  toward  the 
dorsal  surface  of  the  hand  and  does  not  touch  any  por- 
tion of  the  hand ;  this  is  accomplished  by  placing  only 
the  overhanging  portion  of  the  plug  between  the  fingers. 
The  needle  containing  the  bit  of  colony  is  now  to  be 
thrust  into  the  medium  in  the  tube  if  a  stab-culture  is 
desired,  or  rubbed  gently  over  its  surface  if  a  smear- 
culture  is  to  be  made.  The  needle  is  then  Avithdrawn, 
the  cotton  plug  replaced,  and  the  needle  sterilized  before  it 
is  laid  down.  Neither  the  needle  nor  its  handle  should 
touch  the  inner  sides  of  the  test-tube  if  it  can  be  avoided. 
The  tube  is  then  labelled  and  set  aside  for  observa- 
tion. The  growth  which  appears  in  the  tube  after 
twenty-four  to  thirty-six  hours  will  be  a  pure  culture 
of  the  organisms  of  which  the  colony  was  composed. 


148 


BACTERIOLOGY. 


Cultures  of  this  form  are  not  only  useful  as  a  means 
of  preserving  the  different  organisms  with  which  we 
may  be  working,  but  serve  also  to  bring  out  certain 


FIG.  34. 


Series  of  stab-cultures  in  gelatin,  showing  modes  of  growth  of  different 
species  of  bacteria. 

characteristics  of   different  organisms  when  grown  in 
this  way. 

If  gelatin  be  employed  and  the  organism  which  has 
been  introduced  into  it  possesses  the  power  of  bringing 
about  liquefaction,  it  will  soon  be  discovered  that  the 


TEST-TUBE,  STAB-  AND  SMEAR-CULTURES.    149 

mode  of  liquefaction  differs  with  different  organisms. 
Some  bacteria  cause  a  liquefaction  which  spreads  across 
the  whole  upper  surface  of  the  gelatin  and  continues 
gradually  downward ;  with  others  it  occurs  in  a  funnel- 
shape,  the  broad  end  of  the  funnel  being  uppermost  and 
the  point  downward,  corresponding  to  the  track  of  the 
needle ;  at  times  a  stocking-  or  sac-like  liquefaction 
may  be  noticed.  (See  Fig.  34.) 

NOTE. — Obtain  a  number  of  organisms  from  differ- 
ent sources  in  pure  cultures  by  the  method  given.  Plant 
them  as  pure  cultures,  all  at  the  same  time,  in  gelatin — 
preferably  gelatin  of  the  same  making — retain  them 
under  the  same  conditions  of  temperature,  and  sketch 
the  finer  differences  in  the  way  in  which  liquefaction 
occurs. 


CHAPTER  X. 

Methods  of  staining — Solutions  employed — Preparation  and  staining  of 
cover-slips— Preparation  of  tissues  for  section-cutting — Staining  of 
tissues — Special  stain  ing-methods. 

A  COMPLETE  list  of  solutions  and  methods  that  are 
recommended  for  the  staining  of  bacteria  is  not  essen- 
tial to  the  work  of  the  beginner,  so  that  only  those 
which  are  of  the  most  common  application  will  be  given 
in  this  book.  In  general,  it  suffices  to  say  that  bac- 
teria stain  best  with  watery  solutions  of  the  basic  ani- 
line dyes,  and  of  these,  fuchsin,  gentian- violet,  and 
methylene-blue  are  those  most  frequently  employed. 

In  practical  work  bacteria  are  either  dried  upon  cover- 
slips  and  then  stained,  or  stained  in  sections  of  tissues  in 
which  they  have  been  deposited  during  the  course  of 
disease.  In  both  processes  the  essential  point  to  be  borne 
in  mind  is  that  the  bacteria,  because  of  their  microscopic 
dimensions,  require  to  be  more  conspicuously  stained 
than  the  surrounding  materials  upon  the  cover-slips  or 
in  the  sections,  otherwise  their  recognition  is  a  matter  of 
the  greatest  difficulty,  if  not  of  impossibility.  For  this 
reason,  especially  in  section-staining,  it  frequently  be- 
comes necessary  to  decolorize  the  tissues  after  removing 
them  from  the  staining-solutions,  in  order  to  render  the 
bacteria  more  prominent,  and  for  this  purpose  special 
methods,  which  provide  for  decolorization  of  the  tissues 
without  robbing  the  bacteria  of  their  color,  are  employed. 
The  ordinary  method  of  cover-slip  examination  of  bac- 

150 


COVER-SLIP  PREPARATIONS.  151 

teria,  constantly  in  use  in  these  studies,  is  performed  in 
the  following  way : 

COVER-SLIP  PREPARATIONS. — In  order  that  the  dis- 
tribution of  the  organisms  upon  the  cover-slips  may 
be  uniform  and  in  as  thin  a  layer  as  possible  it  is  es- 
sential that  the  slips  should  be  clean  and  free  from 
grease.  For  cleansing  the  slips  several  methods  may 
be  employed. 

The  simplest  plan  with  new  cover-slips  is  to  immerse 
them  for  a  few  hours  in  strong  nitric  acid,  after  which 
they  are  rinsed  in  water,  then  in  alcohol,  then  ether, 
and,  finally,  they  may  be  kept  in  alcohol  to  which  a 
little  ammonia  has  been  added.  When  about  to  be 
used  they  should  be  wiped  dry  with  a  clean  cotton  or 
silk  handkerchief. 

If  the  slips  have  been  previously  usedj  boiling  in 
strong  soap  solution,  followed  by  rinsing  in  clean  warm 
water,  and  then  treating  as  above,  renders  them  clean 
enough  for  ordinary  purposes. 

A  method  commonly  employed  is  to  remove  all  coarse 
adherent  matter  from  slips  and  slides  by  allowing  them 
to  remain  for  a  time  in  strong  nitric  or  sulphuric  acid. 
They  are  removed  from  the  acid  after  several  days, 
rinsed  in  water,  and  treated  as  above.  Knauer  has  re- 
cently suggested  the  boiling  of  soiled  cover-slips  and 
slides  for  from  twenty  to  thirty  minutes  in  a  10  per  cent, 
watery  solution  of  lysol,  after  which  they  are  to  be  care- 
fully rinsed  in  water  until  all  trace  of  the  lysol  has  dis- 
appeared. They  are  then  to  be  wiped  dry  with  a  clean 
handkerchief. 

Loffler's  method,  which  provides  for  the  complete 
removal  of  all  grease,  is  to  warm  the  cover-slips  in  con- 
centrated sulphuric  acid  for  a  time  and  then  rinse  them 


152  BACTERIOLOGY. 

in  water,  after  which  they  are  kept  in  a  mixture  of 
equal  parts  of  alcohol  and  ammonia.  They  are  to  be 
dried  on  a  cloth  from  which  all  fat  has  been  extracted. 
Steps  in  making  the  preparations.  Place  upon  the 
centre  of  one  of  the  clean,  dry  cover-slips  a  very  small 
drop  of  distilled  water  or  physiological  salt-solution. 
With  a  platinum  needle,  which  has  been  sterilized  in 
a  gas-flame  just  before  using  and  allowed  to  cool,  take 
up  a  very  small  portion  of  the  colony  to  be  examined 
and  mix  it  carefully  with  the  drop  on  the  slip  until 
there  exists  a  very  thin  homogeneous  film  over  the 
larger  part  of  the  surface.  This  is  to  be  dried  upon 
the  slip  by  either  allowing  it  to  remain  upon  the  table 
in  the  horizontal  position  under  a  cover,  to  protect  it 
from  dust,  or  by  holding  it  between  the  fingers  (not  with 
forceps),  at  some  distance  above  a  gas-flame,  until  it 
is  quite  dry.  If  held  with  the  forceps  over  the  flame 
at  this  stage,  too  much  heat  may  be  unconsciously  ap- 
plied, and  the  morphology  of  the  organisms  in  the  prep- 
aration distorted.  When  held  between  the  fingers 
with  the  thin  layer  of  bacteria  away  from  the  flame  no 
such  accident  is  likely  to  occur.  When  the  whole 
pellicle  is  completely  dried  the  slip  is  to  be  taken  up 
with  forceps,  and,  holding  the  side  upon  which  the  bac- 
teria are  deposited  away  from  the  direct  action  of  the 
flame,  it  is  to  be  passed  through  the  flame  three  times, 
a  little  more  than  one  second  being  allowed  for  each 
transit.  Unless  the  preliminary  drying  at  the  low  tem- 
perature has  been  complete,  the  preparation  will  be 
rendered  worthless  by  the  subsequent  "  fixing "  at  the 
higher  temperature,  for  the  reason  that  the  protoplasm 
of  bacteria  when  moist  coagulates  at  these  tempera- 
tures, and  in  doing  so  the  normal  outline  of  the  cells  is 


COVER-SLIP  PREPARATIONS.  153 

altered.  If  carefully  dried  before  fixing,  this  does  not 
occur  and  the  morphology  of  the  organism  remains  un- 
changed. 

A  better  plan  for  the  process  of  fixing  is  to  employ 
a  copper  plate  about  35  cm.  long  by  10  cm.  wide  by 
0.3  cm.  thick.  This  plate  is  laid  upon  an  iron  tripod 
and  a  small  gas-flame  is  placed  beneath  one  of  its 
extremities.  By  this  arrangement  one  can  get  a  gradu- 
ated temperature,  beginning  at  the  part  of  the  plate 
above  the  gas-flame  where  it  is  hottest,  and  becom- 
ing gradually  cooler  toward  the  other  end  of  the  plate, 
which  may  be  of  a  very  low  temperature.  By  dropping 
water  upon  the  plate,  beginning  at  the  hottest  point  and 
proceeding  toward  the  cooler  end,  it  is  easy  to  determine 
the  point  at  which  the  water  just  boils ;  it  is  at  a  little 
below  this  point  that  the  cover-slips  are  to  be  placed, 
bacteria  side  up,  and  allowed  to  remain  about  ten  min- 
utes, when  the  fixing  will  be  complete.  The  same  may 
be  accomplished  in  a  small  copper  drying-oven,  which 
is  regulated  to  remain  at  a  temperature  of  from  95°  to 
98°  C.  In  very  particular  work  this  plan  is  to  be  pre- 
ferred to  the  process  of  passing  the  cover- slips  through 
a  flame,  as  the  organisms  are  always  subjected  to  the 
same  degree  of  heat,  and  the  distortions  which  some- 
times* occur  from  too  great  and  irregular  application  of 
high  temperatures  may  be  eliminated.  The  fixing  con- 
sists in  drying  or  coagulating  the  gelatinous  envelope  sur- 
rounding the  organisms,  by  which  means  they  are  caused 
to  adhere  to  the  surface  of  the  cover-slip.  It  is  some- 
times desirable  to  fix  the  preparation  without  the  use 
of  heat,  as  in  the  case  of  pus  or  other  exudates.  In 
this  event,  after  drying  the  thinly  spread  material  care- 
fully in  the  air,  the  cover-slip  on  which  it  is  placed  is 


154  BACTERIOLOGY. 

immersed  in  a  mixture  of  equal  parts  of  absolute  alco- 
hol and  ether  for  about  15  minutes.  At  the  end  of 
this  time  it  may  be  removed  and  stained.  The  advan- 
tage of  this  method  is  that  there  is  less  distortion  and, 
as  a  rule,  less  precipitation  (or,  perhaps  better,  no  char- 
ring) of  extraneous  matter.  When  fixed,  staining  is 
usually  a  simple  matter.  The  majority  of  bacteria  with 
which  the  beginner  will  have  to  deal  stain  readily  with 
watery  solutions  of  any  of  the  basic  aniline  dyes,  such, 
for  instance,  as  fuchsin,  methylene-blue,  or  gentian- 
violet. 

To  stain  the  fixed  cover-slip  preparation,  it  is  taken 
by  one  of  its  edges  between  forceps,  and  a  few  drops 
of  a  watery  solution  of  either  of  the  dyes  named  are 
placed  upon  the  film  and  allowed  to  remain  twenty 
to  thirty  seconds.  The  slip  is  then  carefully  rinsed 
in  water,  and  without  drying  is  placed  bacteria  down 
upon  a  slide ;  the  excess  of  water  is  taken  up  by 
covering  it  with  blotting-paper  and  gently  pressing 
upon  it,  after  which  the  preparation  is  ready  for  ex- 
amination. 

Another  plan  sometimes  used  is  to  bring  the  slip 
upon  the  slide,  bacteria  down,  without  rinsing  oif  the 
staining-fluid  ;  the  excess  of  fluid  is  removed  with 
blotting-paper  and  the  preparation  is  ready  for  exam- 
ination with  the  microscope.  This  method  is  satis- 
factory and  time-saving,  but  must  always  be  prac- 
tised with  care.  The  staining-fluid  should  always  be 
filtered  before  using,  to  rid  it  of  insoluble  particles 
which  might  be  taken  for  bacteria.  If  upon  exami- 
nation the  preparation  proves  of  particular  interest, 
so  that  it  is  desirable  to  preserve  it,  then  it  may  be 
mounted  permanently.  The  drop  of  immersion  oil 


COVER-SLIP  PREPARATIONS.  155 

is  to  be  removed  from  the  surface  of  the  slip  with 
blotting-paper,  and  the  slip  loosened,  or  rather  floated, 
from  the  slide  by  allowing  water  to  flow  around  its 
edges.  It  is  then  taken  up  with  forceps,  carefully  de- 
prived of  the  water  adhering  to  it  by  means  of  blotting- 
paper,  and  allowed  to  dry.  When  dry  it  is  mounted  in 
x\  lol-Canada-balsam  by  placing  a  small  drop  of  the 
balsam  upon  the  surface  of  the  film,  and  then  inverting 
the  slip  upon  a  clean  glass  slide.  It  is  sometimes  de- 
sirable to  have  the  balsam  harden  quickly,  and  a  method 
that  is  commonly  employed  to  induce  this  is  as  follows : 
the  slide,  held  by  one  of  its  ends  between  the  fingers,  is 
warmed  over  a  gas-flame  until  quite  hot;  a  drop  of 
balsam  is  then  placed  on  the  centre  of  it,  and  it  is  again 
wanned ;  the  cover-slip  is  then  placed  in  position,  and 
when  the  balsam  is  evenly  distributed  the  temperature 
is  rapidly  reduced  by  rubbing  the  bottom  of  the  slide 
with  a  towel  wet  with  cold  water.  Usually  the  prepara- 
tion is  firmly  fixed  after  this  treatment ;  a  little  practice 
is  necessary,  however,  in  order  not  to  overheat  and 
crack  the  slide.  The  method  is  applicable  only  to 
cover-slip  preparations,  and  cannot  be  safely  used  with 
tissues. 

IMPRESSION  COVER-SLIP  PREPARATIONS. — Impres- 
sion preparations  differ  from  ordinary  cover-slip  prep- 
arations in  only  one  respect :  they  present  an  impression 
of  the  organisms  as  they  were  arranged  in  the  colony 
from  which  the  preparation  is  made.  They  are  made 
by  gently  covering  the  colony  with  a  thin,  clean  cover- 
slip,  lightly  pressing  upon  it,  and,  without  moving  the 
slip  laterally,  lifting  it  by  one  of  its  edges.  The  organ- 
isms adhere  to  the  slip  in  the  same  relation  to  one 
another  that  they  had  in  the  colony.  The  subsequent 


156  BACTERIOLOGY. 

steps  of  drying,  fixing,  staining,  and  mounting  are  the 
same  as  those  just  given  for  ordinary  cover-slip  prep- 
arations. 

By  this  method  constancies  in  the  arrangement  and 
grouping  of  the  individuals  in  a  colony  can  often  be 
made  out.  Some  will  always  appear  irregularly  massed, 
others  show  growth  in  parallel  bundles,  while  others, 
again,  will  be  seen  as  long,  twisted  threads. 

NOTE. — From  a  colony  of  bacillus  subtilis  make  a 
cover-slip  preparation  in  the  ordinary  way  ;  now  make 
an  impression  cover-slip  preparation  of  another  colony 
of  the  same  organism.  Compare  the  results. 

ORDINARY  STAINING-SOLUTIONS.  —  The  solutions 
commonly  employed  in  staining  cover-slip  preparations 
are,  as  has  been  stated,  watery  solutions  of  the  basic 
aniline  dyes — fuchsin,  gentian- violet,  and  methylene- 
blue.  These  solutions  may  be  made  either  by  directly 
dissolving  the  dyes  in  substance  in  water  until  the 
proper  degree  of  concentration  has  been  reached,  or  by 
using  concentrated  watery  or  alcoholic  solutions  of  the 
dyes  which  may  be  kept  on  hand  as  stock.  The  latter 
method  is  the  one  commonly  practised. 

The  solutions  of  the  colors  which  are  in  constant  use 
in  staining  are  prepared  as  follows : 

Prepare  as  stock,  saturated  alcoholic  or  watery  solu- 
tions of  fuchsin,  gentian-violet,  and  methylene-blue. 
These  solutions  are  best  made  by  pouring  into  clean 
bottles  enough  of  the  dyes  in  substance  to  fill  them  to 
about  one-fourth  of  their  capacity.  Each  bottle  should 
then  be  filled  with  alcohol  or  with  water,  tightly  corked, 
well  shaken,  and  allowed  to  stand  for  twenty-four  hours. 


ORDINARY  STAINING-SOLUTIONS.  157 

If  by  then  all  the  staining-material  has  been  dissolved, 
more  should  be  added,  the  bottle  being  again  shaken 
and  allowed  to  stand  for  another  twenty-four  hours; 
this  must  be  repeated  until  a  permanent  sediment  of 
undissolved  color  ing- matter  is  seen  upon  the  bottom 
of  the  bottle.  The  bottles  are  then  to  be  labelled 
"  saturated  alcoholic "  or  "  watery  "  solution  of  fuch- 
sin,  gentian-violet,  or  methylene-blue,  as  the  case  may 
be.  The  alcoholic  solutions  are  not  directly  employed  for 
sta  ining-purposes. 

The  solutions  with  which  staining  is  accomplished 
are  made  from  the  stock  solutions  in  the  following 
way : 

An  ordinary  test-tube  of  about  13  mm.  diameter  is 
three-fourths  filled  with  distilled  water  and  the  con- 
centrated alcoholic  or  watery  solution  of  the  dye 
added,  little  by  little,  until  one  can  just  see  through 
the  solution.  It  is  then  ready  for  use.  Care  must  be 
taken  that  the  color  does  not  become  too  dense.  The 
best  results  are  obtained  when  this  dilution  is  just 

FIG.  35. 


Rack  of  bottles  for  staining-solutions. 

transparent  as  viewed  through   a  layer   about    12   to 
14  mm.  thick. 


158  BACTERIOLOGY. 

The  above  represent  the  staining-solutions  in  every- 
day use.  They  are  kept  in  bottles  supplied  with  stoppers 
and  pipettes  (Fig.  35),  and  when  used  are  dropped  upon 
the  preparation  to  be  stained. 

For  certain  bacteria  which  stain  only  imperfectly 
with  these  simple  solutions  it  is  necessary  to  employ 
some  agent  that  will  increase  the  penetrating  action  of 
the  dyes.  Experience  has  taught  us  that  this  can  be 
accomplished  by  the  addition  to  the  solutions  of  small 
quantities  of  alkaline  substances,  or  by  dissolving  the 
staining-materials  in  strong  watery  solutions  of  either 
aniline  oil  or  carbolic  acid,  instead  of  water — in  other 
words,  by  employing  special  solvents  and  mordants 
with  the  stains. 

Of  the  solutions  thus  prepared  which  may  always  be 
employed  upon  bacteria  that  show  a  tendency  to  stain 
imperfectly,  there  are  three  in  common  use — Loffler's 
alkaline  methylene-blue  solution ;  the  Koch-Ehrlich 
aniline-water  solution  of  either  fuchsin,  gentian-violet, 
or  methylene-blue  ;  and  ZiehPs  solution  of  fuchsin  in 
carbolic  acid.  These  solutions  are  as  follows  : 

Loffler's  alkaline  methylene-blue  solution  : 

Concentrated  alcoholic  solution  of  methylene-blue  .    .      30  c.c. 
Caustic  potash  in  1 : 10,000  solution 100  c.c. 

JKbch-Ehrlich  aniline-water  solution.  To  about  100 
c.c.  of  distilled  water  aniline  oil  is  added  drop  by  drop 
until  the  solution  has  an  opaque  appearance,  the  vessel 
containing  the  solution  being  thoroughly  shaken  after 
the  addition  of  each  drop.  It  is  then  filtered  through 
moistened  filter-paper  until  the  filtrate  is  perfectly 
clear.  To  100  c.c.  of  the  clear  filtrate  add  10  c.c.  of 
absolute  alcohol  and  11  c.c.  of  the  concentrated  alco- 


ORDINARY  STAINING-SOLUTIONS.  159 

holic  solution  of  either  fuchsin,  methy  lone-blue,  or  gen- 
tian-violet, preferably  fuchsin  or  gentian-violet. 
ZiehUs  carbol-fuchsin  solution : 

Distilled  water 100  c.c. 

Carbolic  acid  (crystallized) 5  grammes. 

Alcohol ." 10  c  c 

Fuchsin  in  substance  ......   .   /  .'  .   .  i  gramme. 

Or  it  may  be  prepared  by  adding  to  a  5  per  cent, 
watery  solution  of  carbolic  acid  the  saturated  alcoholic 
solution  of  fuchsin  until  a  metallic  lustre  appears  on 
the  surface  of  the  fluid. 

The  Koch-Ehrlich  solution  decomposes  after  a  time, 
so  that  it  is  better  to  prepare  it  fresh  in  small  quantities 
when  needed  than  to  employ  old  solutions.  Solutions 
older  than  fourteen  days  should  not  be  used. 

The  three  solutions  just  given  may  be  used  for  cover- 
glass  preparations  in  the  ordinary  way. 

In  some  manipulations  it  becomes  necessary  to  stain 
the  bacteria  very  intensely,  so  that  they  may  retain 
their  color  when  exposed  to  the  action  of  decolorizing 
agents.  These  methods  are  usually  employed  when  it 
is  desirable  to  deprive  surrounding  objects  or  tissues  of 
their  color,  in  order  that  the  stained  bacteria  may  stand 
out  in  greater  contrast.  It  is  in  these  cases  that  the 
staining-solution  with  which  the  bacteria  are  being 
treated  is  to  be  warmed,  and  in  some  cases  boiled,  so  as 
further  to  increase  its  penetrating  action.  When  so 
treated,  certain  of  the  bacteria  will  retain  their  color, 
even  when  exposed  to  very  strong  decolorizers.  The 
tubercle  bacillus  is  distinguished  from  the  great  ma- 
jority of  other  bacteria  by  the  tenacity  with  which  it 
retains  the  color  when  treated  in  this  way  ;  it  is  an 


160  BACTERIOLOGY. 

organism  difficult  to  stain,  but  when  once  stained  is 
equally  difficult  to  rob  of  its  color. 

METHOD  OF  STAINING  THE  TUBERCLE  BACILLUS. 
— Select  from  the  sputum  of  a  tuberculous  subject  one 
of  the  small,  white,  cheesy  masses  which  it  is  seen 
to  contain.  Spread  this  upon  a  cover-slip,  dry  and 
fix  it  in  the  usual  way.  The  slip  is  now  to  be  taken 
by  its  edge  with  forceps  and  the  film  covered  with  a 
few  drops  of  either  the  solution  of  Koch-Ehrlich  or 
that  of  Ziehl.  It  is  then  held  over  a  gas-flame,  at  first 
some  distance  away,  gradually  being  brought  nearer 
until  the  fluid  begins  to  boil.  After  it  has  bubbled 
once  or  twice  it  is  removed  from  the  flame,  the  excess 
of  stain  washed  away  in  a  stream  of  water,  then  im- 
mersed in  a  30  per  cent,  solution  of  nitric  acid  in 
water,  and  allowed  to  remain  until  all  color  has  dis- 
appeared. This  takes  longer  in  some  cases  than  in 
others.  One  can  always  determine  if  decolonization  is 
complete  by  washing  off  the  acid  in  a  stream  of  water. 
If  the  preparation  is  still  distinctly  colored,  it  should 
be  again  immersed  in  the  acid ;  if  of  only  a  very  faint 
color,  it  may  be  dipped  in  alcohol,  again  washed  in 
water,  and  stained  with  some  contrast-color.  If,  for 
example,  the  tubercle  bacilli  have  been  stained  with 
fuchsin,  methylene-blue  forms  a  good  contrast-stain. 
In  making  the  contrast-stain  the  steps  in  the  process 
are  exactly  those  followed  in  the  ordinary  staining 
of  cover-slip  preparations  in  general :  the  slip  contain- 
ing the  stained  tubercle  bacilli  is  carefully  rinsed  in 
water,  and  a  few  drops  of  the  methylene-blue  solution 
placed  upon  it  and  allowed  to  remain  for  thirty  or 
forty  seconds,  when  it  is  again  rinsed  in  water  and  ex- 
amined microscopically.  For  the  purpose  of  observ- 


STAINING   THE  TUBERCLE  BACILLUS.        161 

ing  the  difference  in  behavior  of  the  tubercle  bacilli 
and  the  other  organisms  present  in  the  preparation 
toward  this  method  of  staining,  it  is  well  to  exam- 
ine the  preparation  microscopically  before  the  con- 
trast-stain is  made;  then  give  it  the  contrast-color, 
and  again  examine.  It  will  be  seen  that  before  the 
contrast-color  has  been  given  to  the  preparation  the 
tubercle  bacilli  are  the  only  stained  objects  to  be 
made  out,  and  the  preparation  appears  devoid  of 
other  organisms;  but  upon  examining  it  after  it  has 
received  the  contrast-color  a  great  many  other  or- 
ganisms will  appear;  these  take  on  the  second  color 
employed,  while  the  tubercle  bacilli  retain  their  orig- 
inal color.  Before  decolorization  all  organisms  in  the 
preparation  were  of  the  same  color,  but  during  the  appli- 
cation of  the  decolorizing  solution  all  except  the  tubercle 
bacilli  gave  up  their  color.  This  micro-chemical  charac- 
teristic, together  with  reactions  to  be  described,  serves 
to  differentiate  the  tubercle  bacillus  from  other  organ- 
isms with  which  it  might  be  confounded.  A  number 
of  different  methods  have  been  suggested  for  the  stain- 
ing of  tubercle  bacilli,  but  the  original  method  as  em- 
ployed by  Koch  is  so  satisfactory  in  its  results  that  it  is 
not  advisable  to  substitute  others  for  it.  The  above 
differs  from  the  original  Koch-Ehrlich  method  for  the 
staining  of  tubercle  bacilli  in  sputum  only  in  the  occa- 
sional employment  of  ZiehPs  carbol-fuchsin  solution 
and  in  the  method  of  heating  the  preparation  with  the 
stain  ing-fluid  upon  it. 

As  Nuttall  has  pointed  out,  however,  the  strong  acid 
decolorizer  used  in  this  method  can,  with  advantage, 
be  replaced  by  much  more  dilute  solutions,  as  a  num- 
ber of  the  bacilli  are  entirely  decolorized  by  the  too 
11 


162  BACTERIOLOGY. 

energetic  action  of  the  strong  acids.  He  recommends 
the  following  method  of  decolorization  :  after  staining 
the  slip  or  section  in  the  usual  way,  pass  it  through 
three  alcohols ;  it  is  then  to  be  washed  in  a  solution 
composed  of 

Water 150  c.c. 

Alcohol 50  c.c. 

Concentrated  sulphuric  acid 20  to  30  drops. 

From  this  it  is  removed  to  water  and  carefully  rinsed. 
The  remaining  steps  in  the  process  are  the  same  as  those 
given  in  the  other  methods. 

GABBETT'S  METHOD  for  the  staining  of  tubercle 
bacilli  recommends  itself  because  of  its  simplicity  and 
the  rapidity  with  which  it  can  be  performed.  By  many 
it  is  considered  the  best  method  for  routine  employ- 
ment. It  consists  in  staining  the  cover-slips,  prepared 
in  the  manner  given,  for  from  two  to  five  minutes  in 
a  cold  carbol-fuchsin  solution,  after  which  they  are  sub- 
jected to  the  action  of  Gabbett's  methylene-blue  sul- 
phuric acid  solution.  This  latter  consists  of 

Sulphuric  acid  (strength  25  per  cent.)    ....  100  c.c. 
Methylene-blue,  in  substance 1  to  2  grammes. 

The  cover-slips  are  then  rinsed  in  water  and  are  ready 
for  examination.  The  tubercle  bacilli  will  be  stained 
red  by  the  fuchsin,  while  all  other  bacteria,  cell-nuclei, 
etc.,  will  be  tinted  blue. 

GRAM'S  METHOD. — Another  important  differential 
method  of  staining  which  is  very  commonly  employed 
is  that  recommended  by  Gram.  In  this  method  the 
objects  are  treated  with  an  aniline-water  solution  of 
gentian-violet  made  after  the  formula  of  Koch-Ehrlich. 


GLACIAL  ACETIC  ACID  METHOD.  163 

After  remaining  in  this  for  twenty  to  thirty  minutes 
they  are  immersed  in  a  solution  composed  of 

Iodine 1  gramme. 

Potassium  iodide    ..............       2  grammes. 

Distilled  water    ...............    300  c.c. 

In  this  they  remain  for  about  five  minutes ;  they  are 
then  transferred  to  alcohol  and  thoroughly  rinsed.  If 
still  of  a  violet  color,  they  are  again  treated  with  the 
iodine  solution,  followed  by  alcohol,  and  this  is  con- 
tinued until  no  trace  of  violet  is  visible  to  the  naked 
eye.  They  may  then  be  examined,  or  a  contrast-color 
of  carmine  or  Bismarck-brown  may  be  given  them. 

This  method  is  particularly  useful  in  demonstrating 
the  capsule  which  is  seen  to  surround  some  bacteria, 
especially  micrococcus  lanceolatus  of  pneumonia. 

GLACIAL  ACETIC  ACID  METHOD. — Another  method 
that  may  be  employed  for  demonstrating  the  presence 
of  the  capsule  surrounding  certain  organisms  is  to  pre- 
pare the  cover-slips  in  the  ordinary  way,  then  cover  the 
layer  of  bacteria  upon  them  with  glacial  acetic  acid, 
which  is  instantly  poured  off  (not  washed  off  in  water), 
and  the  aniline-water  gentian-violet  solution  dropped 
upon  them ;  this  is  allowed  to  remain  three  or  four 
minutes,  is  poured  off,  and  a  few  drops  more  are  added, 
and  lastly  the  slip  is  washed  in  a  solution  of  sodium 
chloride.  Usually  this  is  of  the  strength  of  physio- 
logical salt-solution,  viz.,  0.6  to  0.7  per  cent. ;  but  at 
times  it  must  be  stronger,  occasionally  as  concen- 
trated as  1.5  to  2  per  cent.  The  reason  for  this 
is  that  if  the  slips  be  washed  in  water,  or  in  salt- 
solution  that  is  too  weak,  the  mucin  capsule  that 
has  been  coagulated  by  the  acetic  acid  is  redissolved 


164  BACTERIOLOGY. 

and  rendered  invisible.  This  does  not  occur  when  the 
salt-solution  is  of  the  proper  strength — a  point  that  can 
be  determined  only  after  a  few  trials  with  solutions  of 
different  strengths.  (Welch.)  A  very  clear,  sharply  cut 
picture  usually  follows  this  method  of  procedure. 

Ribbert  also  recommends  for  the  staining  of  capsu- 
lated  bacteria  the  momentary  immersion  of  the  cover- 
slips  in  a  saturated  solution  of  dahlia  in  a  mixture  of 
100  parts  of  water,  50  parts  of  alcohol,  and  12J  parts 
of  glacial  acetic  acid;  after  which  the  excess  of  color 
is  removed  by  washing  in  water. 

STAINING  OF  SPORES. — We  have  learned  that  one  of 
the  points  by  which  spores  may  be  recognized  is  their 
refusal  to  take  up  stainiug-substances  when  applied  in 
the  ordinary  way.  They  may,  however,  be  stained  by 
special  methods ;  of  these,  one  that  has  given  fairly  satis- 
factory results  in  our  hands  is  as  follows :  the  cover- 
slip  is  to  be  prepared  from  the  material  containing  the 
spores  in  the  ordinary  way,  dried,  and  fixed.  It  is  then 
to  be  held  by  its  edge  with  forceps,  and  its  surface 
covered  with  Loffler's  alkaline  methylene-blue  solu- 
tion. It  is  then  held  over  the  Bunsen  flame  until  the 
fluid  boils ;  it  is  then  removed,  and  after  a  few  seconds 
is  heated  again.  This  is  continued  for  about  one  min- 
ute, after  which  it  is  washed  in  water  and  then  de- 
colorized in 

Alcohol  (80  per  cent.)     98  c.c. 

Nitric  acid ."....     2  c.c. 

until  all  visible  blue  color  has  disappeared.  It  is  then 
rinsed  in  water  and  dipped  for  from  3  to  5  seconds  in 

Saturated  alcoholic  solution  of  eosin 10  c.c. 

Water      .   .  .    90  c.c. 


MOELLER'S  METHOD  FOR  STAINING  SPORES.    165 

after  which  it  is  again  rinsed  in  water  and  finally 
mounted  for  examination.  If  the  decolorization  in 
the  acid  alcohol  be  not  carried  too  far,  the  preparation 
will  show  the  spores  stained  blue  and  the  bodies  of  the 
cells  to  have  taken  on  the  rose  color  characteristic  of  eosin. 
By  another  process  the  cover-slip  is  floated,  bacteria 
down,  upon  the  surface  of  freshly  prepared  Koch- 
Ehrlich  solution  of  fuchsin  contained  in  a  watch-crys- 
tal. This  is  then  held  by  its  edge  with  forceps  about 
2  cm.  above  a  very  small  flame  of  a  Bunsen  burner, 
care  being  taken  that  the  flame  touches  only  the  centre 
of  the  bottom  of  the  crystal.  After  a  few  seconds  the 
crystal  is  gradually  elevated  until  it  is  about  6  to  8  cm. 
above  the  flame ;  then  it  is  slowly  moved  down  to  the 
flame  again,  and  this  up-and-down  movement  is  con- 
tinued until  the  staining-fluid  begins  to  boil.  As  soon 
as  a  few  bubbles  have  been  given  off  it  is  held  aside 
for  a  minute  or  two,  when  the  heating  is  repeated. 
When  the  boiling  begins  the  crystal  is  again  held  aside 
for  a  minute  or  two.  The  crystal  is  heated  in  this  way 
five  or  six  times.  When  the  fluid  has  stood  for  about  five 
minutes  after  the  last  boiling  the  preparation  is  trans- 
ferred, without  washing  in  water,  to  a  second  watch- 
crystal  containing  the  following  decolorizing  solution  : 

Absolute  alcohol 100  c.c. 

Hydrochloric  acid 3  c.c. 

In  this  solution  it  is  placed,  bacteria  up,  and  the 
vessel  is  tilted  from  side  to  side  for  about  one  minute. 
It  is  then  removed,  washed  in  water,  and  stained  with 
the  methylene-blue  solution.  The  spores  will  be  stained 
red  and  the  body  of  the  cells  blue. 

MOELLER'S  METHOD   FOR  STAINING  SPORES. — A 


166  BACTERIOLOGY. 

method  that  has  recently  been  published  by  Moeller 
is  designed  to  favor  the  penetration  of  the  coloring- 
material  through  the  spore-membrane  by  macerating 
the  spores  in  a  solution  of  chromic  acid  before  staining 
them.  It  is  as  follows  : 

The  cover-slips  are  prepared  in  the  usual  way,  or  the 
fixing  may  be  accomplished  with  absolute  alcohol  in- 
stead of  high  temperatures.  The  preparation  is  held 
for  two  minutes  in  chloroform,  washed  in  water,  placed 
for  from  one-half  to  two  minutes  in  a  5  per  cent,  solu- 
tion of  chromic  acid,  again  washed  in  water,  and  stained 
with  carbol-fuchsin.  In  the  process  of  staining,  the  slip 
is  taken  by  the  corner  with  forceps,  and  carbol-fuchsin 
is  dropped  upon  the  side  containing  the  spores.  It  is 
then  held  over  a  flame  until  it  boils,  and  then  held 
some  distance  above  the  flame  for  one  minute.  The 
staining-fluid  is  then  poured  off  and  the  preparation  is 
completely  decolorized  in  5  per  cent,  sulphuric  acid, 
again  washed  in  water,  and  finally  stained  for  thirty 
seconds  in  the  watery  methyl  ene-blue  solution.  The 
spores  will  be  red,  the  body  of  the  cells  blue. 

In  this  method  the  object  of  the  preliminary  ex- 
posure to  chloroform  is  to  dissolve  any  crystals  of  leci- 
thin, cholesterin,  or  fat  that  may  be  in  the  preparation, 
and  which  when  stained  might  cause  confusion. 

It  must  be  remembered  that  there  are  conspicuous 
differences  in  the  behavior  of  spores  of  different  bacteria 
to  staining-methods  and  of  the  spores  of  a  single  species 
in  different  stages  of  development.  Some  stain  readily 
by  either  of  the  methods  especially  devised  for  this 
purpose,  while  others  can  hardly  be  stained  at  all,  or 
only  with  the  greatest  difficulty,  by  any  of  the  known 
processes ;  some  stain  readily  when  fully  developed,  but 


(METHODS  FOR  STAINING  FLAGELLA.  167 
with  difficulty  when  only  partly  developed ;  others  have 
this  peculiarity  reversed. 

LOFFLER'S  METHOD  FOR  STAINING  FLAGELLA. — 
For  the  demonstration  of  the  locomotive  apparatus  pos- 
sessed by  motile  bacteria  we  are  indebted  to  Loffler. 
By  a  special  method  of  staining,  in  which  the  use  of 
mordants  played  the  essential  part,  he  has  shown  that 
these  organisms  possess  very  delicate,  hair-like  appen- 
dages, by  the  lashing  movements  of  which  they  propel 
themselves  through  the  fluid  in  which  they  are  growing. 
The  method  as  given  by  Loffler  is  as  follows  : 

It  is  essential  that  the  bacteria  be  evenly  and  not 
too  numerously  distributed  upon  the  cover-slip.  The 
slips  must  therefore  be  perfectly  clean.  (See  Lofflet^s 
method  of  cleaning  cover-slips.)  Five  or  six  of  the 
carefully  cleansed  cover-slips  are  to  be  placed  in  a  line 
on  a  table,  and  on  the  centre  of  each  slip  a  very  small 
drop  of  tap- water  is  placed.  From  the  culture  to  be 
examined  a  minute  portion  is  transferred  to  the  first 
slip  and  carefully  mixed  Avith  the  drop  of  water ;  from 
this  mixture  a  small  portion  is  transferred  to  the  second, 
and  from  the  second  to  the  third  slip,  and  so  on,  in  this 
way  insuring  a  dilution  of  the  number  of  organisms 
present  in  the  preparations.  These  slips  are  then  dried 
and  fixed  in  the  ordinary  way.  They  are  next  to  be 
warmed  in  the  following  solution  : 

Tannic  acid  solution  in  water  (20  acid,  80  water)  ....    10  c.c. 

Cold  saturated  solution  of  ferrous  sulphate 5  c.c. 

Saturated  watery  or  alcoholic  solution  of  fuchsin    ...      1  c.c. 

This  solution  represents  the  mordant.  A  few  drops 
of  it  are  to  be  placed  upon  the  film  of  bacteria  on  the 
cover-slip,  which  is  then  to  be  held  over  a  flame  until 


168  BACTERIOLOGY. 

the  solution  begins  to  steam.  It  should  not  be  boiled. 
After  steaming,  the  mordant  is  washed  off  in  water  and 
finally  in  alcohol.  The  bacteria  are  then  to  be  stained 
in  a  saturated  aniline-water-ftichsin  solution. 

When  treated  in  this  way  different  bacteria  behave 
differently :  the  flagella  of  some  stain  readily  in  the 
above  solutions ;  others  require  the  addition  of  an  alkali 
in  varying  quantities ;  while  others  stain  best  after  the 
addition  of  acids.  To  meet  these  conditions  an  exact 
1  per  cent,  solution  of  caustic  soda  in  water  must  be 
prepared,  and  also  a  solution  of  sulphuric  acid  in  water 
of  such  strength  that  one  cubic  centimetre  will  be  ex- 
actly neutralized  by  one  cubic  centimetre  of  the  alkaline 
solution. 

For  different  bacteria  which  have  been  studied  by 
this  method  Loffler  recommends  the  one  or  the  other  of 
these  solutions  to  be  added  to  the  mordant  in  the  follow- 
ing proportions. 

Of  the  acid  solution  : 

For  spirillum  cholerse  Asiaticse,  i  to  1  drop  of  acid  to  16  c.c.  of  mordant. 
For  spirillum  rubrum,  9  "  "  "  " 

For  spirillum  MetchniJcoffi,  4  "  "  "  " 

For  bacillus  pyocyaneus,  5 

For  spirillum  concentricum,  no  addition  of  either  acid  or  alkali. 

Of  the  alkaline  solution  : 

For  bacillus  mesentericus  vulgatus,  4  drops  of  alkali  to  16  c.c.  of  mordant. 
For  micrococcus  agilis,  20  »«•;'-,«  « 

For  bacillus  typJiosus,  22  "  "  "  " 

For  bacillus  subtilis,  28-30        "  "  "  " 

For  bacillus  cedematis  maligni,        36-37        "  "  "  " 

For  bacillus  anthracis  symp- 


tomatici,  ' 


The  drops  used  run  22  to  the  cubic  centimetre. 
For  other  organisms  one  must  determine  whether  the 
results  are  better  after  the  addition  of  acid  or  alkali, 


BUNGE'S  METHOD.  169 

and  how  much  of  either  is  required.  In  general,  it  may 
be  said  that  bacteria  which  produce  acids  in  the  media 
in  which  they  are  growing  require  the  addition  of  alka- 
lies to  the  mordant,  while  those  that  produce  alkalies 
require  acids  to  be  added.  By  following  Loffler's  direc- 
tions the  delicate,  hair-like  flagella  on  motile  organisms 
may  be  rendered  plainly  visible. 

There  are  several  points  and  slight  modifications  in 
connection  with  this  method  that  require  to  be  empha- 
sized in  order  to  insure  success :  the  culture  to  be  em- 
ployed should  be  young,  not  over  18-20  hours  old;  it 
should  have  developed  for  this  time  on  fresh  agar-agar 
at  37°  to  38°  C. ;  the  mordant  should  not  be  perfectly 
fresh,  as  the  best  results  are  obtained  from  the  use  of 
old  solutions  that  have  stood  exposed  to  the  air  and 
that  have  been  filtered  just  before  using ;  when  placed 
on  the  cover-slip  and  held  over  the  flame  never  heat  the 
mordant  to  the  boiling-point ;  indeed,  the  best  results  are 
obtained  when  the  preparation  is  held  high  above  the  flame 
and  removed,  from  it  at  the  first  evidence  of  vaporization, 
or,  better  still,  a  little  before  this  point  is  reached.  AVe 
have  derived  no  advantage  from  the  addition  of  acids 
or  alkalies  to  the  mordant,  as  recommended  by  Loffler ; 
but  obtain,  with  a  fair  degree  of  regularity,  satisfactory 
results  through  the  use  of  the  neutral  mordant  alone.1 

BUNGE'S  METHOD. — A  useful  modification  of  Lof- 
fler's method  is  that  recommended  by  Bunge  :  prepare 
a  saturated  solution  of  tannin,  and  a  solution  of  liquor 
ferri  sesquichlor.  of  the  strength  of  1  :  20  of  distilled 
water.  To  3  parts  of  the  tannin  solution  add  1  part  of 

1 1  am  indebted  to  Dr.  James  Homer  Wright,  Thomas  Scott  Fellow 
in  Hygiene,  1892-'93,  University  of  Pennsylvania,  for  some  of  the 
suggestions  in  connection  with  the  modification  of  this  method. 


170  BACTERIOLOGY. 

the  dilute  iron  solution.  To  10  c.c.  of  such  a  mixture 
add  1  c.c.  of  concentrated  watery  solution  of  fuchsin. 
This  mordant  is  not  to  be  used  fresh,  but  only  after 
standing  exposed  to  the  air  for  several  days  (better  for 
several  weeks).  After  preparing  the  cover-slip  with  all 
precautions  necessary  to  cleanliness  the  filtered  mordant 
is  allowed  to  act  cold  for  about  five  minutes,  after  which 
it  is  slightly  warmed;  the  slip  is  then  washed  in 
water,  dried,  and  faintly  stained  with  carbol-fuchsin. 
No  addition  of  acid  or  alkali  to  the  mordant  is  neces- 
sary. 

THE  METHOD  OF  VAN  ERMENGEM. — Another 
method  of  demonstrating  the  presence  of  flagella  is  that 
suggested  by  Van  Ermengem.  It  is  somewhat  more 
complicated  than  either  of  the  preceding  methods.  The 
steps  in  the  process  are  as  follows : 

In  the  centre  of  a  perfectly  cleaned  cover-slip  place 
a  drop  of  a  very  dilute  suspension,  in  physiological  salt- 
solution,  of  a  10-  to  18-hour  old  agar-agar  culture  of 
the  organism  to  be  studied.  The  suspension  of  the 
organisms  in  the  salt-solution  should  be  very  dilute  in 
order  to  favor  the  isolation  of  single  cells  on  the  slip 
and  also  to  obviate  the  occurrence  of  excessive  precip- 
itation. The  slips  are  then  to  be  dried  in  the  air  and 
fixed  over  a  gas-flame  in  the  usual  manner. 

The  mordant  used  consists  of: 

Osmic  acid  (2  per  cent,  solution) 1  part. 

Tannin  (10-25  per  cent,  solution) 2  parts. 

To  the  mordant  4  or  5  drops  of  glacial  acetic  acid  may 
be  added,  but  experience  has  shown  this  to  be  hardly 
necessary.  Place  a  drop  or  two  of  the  mordant  on  the 
cover-slip  to  be  stained,  and  allow  it  to  act  for  one-half 


BOWHILL'S  METHOD.  171 

hour  at  room-temperature,  or  for  five  minutes  at  50°  to 
60°  C.  Wash  carefully  in  water  and  in  alcohol,  and  then 
immerse  for  a  few  seconds  in  the  "  sensitizing  bath," 
viz.,  a  0.25-0.5  per  cent,  solution  of  silver  nitrate. 
Without  washing,  bring  the  slip  into  a  watch-crystalful 
of  the  "reducing  and  reinforcing  bath,"  viz.: 

Gallic  acid 5  grammes. 

Tannin 3      " 

Fused  potassium  acetate 10      " 

Distilled  water 350      " 

After  a  few  seconds  pass  the  slip  back  into  a  watch- 
crystal  containing  the  dilute  silver  bath  (0.25-0.5  per 
cent,  solution  of  silver  nitrate  in  water)  and  keep  it 
in  constant  motion  until  the  solution  begins  to  take  on 
a  brown  or  blackish  color.  Wash  in  water  thoroughly  ; 
dry  with  blotting-paper,  and  mount  in  balsam. 

BOWHILL'S  METHOD. — From  a  fresh  culture  on  agar- 
agar  make  a  suspension  in  sterilized  distilled  water  in  a 
test-tube ;  allow  this  to  stand  for  five  minutes ;  from  the 
supernatant  fluid  prepare  cover-slips,  care  being  taken 
that  the  slips  are  clean  and  that  too  much  heat  is  not 
applied  in  fixing;  float  the  cover-slip,  bacteria  down, 
upon  some  of  the  following  mixture  in  a  watch-glass  : 

Saturated  alcoholic  solution  of  orcein  (about  10  days  old)  .  15  c.c. 
20  per  cent,  solution  of  tannic  acid  in  water  (acid  dis- 
solved by  heating) 10  c.c. 

Distilled  water 30  c.c. 

Gently  warm  the  fluid  and  allow  the  preparation  to 
remain  in  it  for  10-15  minutes;  then  rinse  in  water, 
and  finally  mount  and  examine  in  water  or  dry  and 
mount  in  xylol  balsam.1 

1  Bowhill :  Hygienische  Rundschau,  1898,  No.  3,  p.  105. 


172  BACTERIOLOGY. 


STAINING   IN   GENERAL. 

The  physics  of  staining  and  decolonization  is  hardly 
a  subject  to  be  discussed  at  length  in  a  book  of  this 
character;  but,  as  Kiihne  has  pointed  out,  it  may  be 
briefly  said  that  solutions  which  favor  the  production  of 
diffusion-currents  facilitate  intensity  of  staining,  and  by 
a  similar  process  increase  the  energy  of  decolorizing- 
agents.  For  example,  tissues  which  are  transferred 
from  water  into  watery  solutions  of  the  coloring-mat- 
ters are  less  intensely  stained  and  more  easily  decolor- 
ized than  when  transferred  from  alcohol  into  watery 
staining-fluids ;  for  the  same  reason  tissues  stained  in 
watery  solutions  of  the  dyes  do  not  become  decolorized 
so  readily  when  placed  in  water  as  when  placed  in 
alcohol. 

The  diffusion  of  staining-solutions  in  the  protoplasm 
of  dried  bacteria,  as  found  upon  cover-slip  preparations, 
is  much  greater  and  more  rapid  than  when  the  same 
bacteria  are  located  in  the  interstices  of  tissues.  These 
differences  are  not  in  the  bacteria  themselves,  but  in  the 
obstruction  to  diffusion  offered  by  the  tissues  in  which 
they  are  located.  The  result  of  absence  of  diffusion 
may  easily  be  illustrated  : 

Prepare  a  cover-slip  preparation,  dry  it  carefully,  fix 
it,  and,  without  allowing  water  to  get  on  it  from  any 
source,  attempt  to  stain  it  with  a  solution  of  the 
dyes  in  absolute  alcohol,  washing  it  subsequently  with 
absolute  alcohol ;  the  result  is  negative.  The  abso- 
lute alcohol  does  not  possess  the  property  of  diffusing 
into  the  dried  tissues,  and  hence,  as  has  been  stated, 
alcoholic  solutions  of  the  staining-dyes  should  not  be 


DECOLORIZISG-SOL  UTIONS.  1 73 

employed.  The  staining-dyes  should  always  be 
watery.1 

DECOLORIZIXG-SOLUTIONS. — As  regards  the  employ- 
ment of  decolorizing-agents,  it  must  always  be  borne  in 
mind  that  objects  which  are  easily  stained  are  also  easily 
decolorized,  and  those  that  can  be  caused  to  take  up  the 
staining-material  only  with  difficulty  are  also  very  diffi- 
cult to  rob  of  their  color.  The  most  common  decolor- 
izer  in  use  is  probably  alcohol — not  absolute  alcohol, 
but  alcohol  containing  more  OP  less  of  water.  Water 
alone  has  this  property,  but  in  a  much  less  degree  than 
dilute  alcohol.  On  the  other  hand,  a  much  more  ener- 
getic decolorization  than  that  possessed  by  either  alone 
can  be  obtained  by  alternate  exposures  to  alcohol  and 
water.  More  energetic  in  their  decolorizing  action  than 
either  water  or  alcohol  are  solutions  of  the  acids.  They 
appear,  particularly  when  they  are  alcoholic  solutions, 
to  diffuse  rapidly  into  tissues  and  bacteria  and  very 
quickly  extract  the  staining-materials  which  have  been 
deposited  there.  For  this  reason  these  solutions  should 
be  employed  with  much  care. 

Very  dilute  acetic  acid  robs  tissues  and  bacteria  of 
their  stain  with  remarkable  activity;  still  more  ener- 
getic are  solutions  of  the  mineral  acids,  and  particularly, 
as  has  been  said,  when  this  action  is  accompanied  by 
the  decolorizing-properties  of  alcohol. 

The  acid  solutions  commonly  employed  are  : 

Acetic  acid  in  from  0.1  to  5  per  cent,  watery  solution. 

1  In  the  beginning  of  this  chapter  it  was  stated  that  the  saturated 
alcoholic  solutions  of  the  dyes  do  not  serve  as  stains  for  bacteria.  It 
must  be  remembered  that  this  holds  only  when  absolute  alcohol  and 
perfectly  dry  coloring-matters  have  been  used.  If  but  a  small  propor- 
tion of  water  is  present,  the  bacteria  may  be  stained  with  these  solu- 
tions, though  the  results  are,  as  a  rule,  unsatisfactory. 


174  BACTERIOLOGY. 

Nitric  acid  in  from  20  to  30  per  cent,  watery  solu- 
tion. 

Hydrochloric  acid  in  from  1  to  3  per  cent,  solution  in 
alcohol. 

STAINING   OF   BACTERIA   IN   TISSUES. 

In  staining  tissues  for  the  purpose  of  demonstrating 
the  bacteria  which  they  may  contain  a  number  of  points 
must  be  borne  in  mind  :  the  conditions  which  favor  the 
diffusion  of  the  staining-fluids  into  the  bacteria  are  now 
not  so  favorable  to  rapid  staining  as  they  were  when 
the  bacteria  alone  were  present  upon  cover-slips ;  the 
staining  of  tissues,  therefore,  requires  a  longer  exposure 
to  the  dyes  than  does  that  of  cover-slips.  In  tissues, 
too,  there  are  other  substances  beside  the  bacteria  which 
become  stained,  and  these,  unless  robbed  in  whole  or  in 
part  of  their  color,  may  so  mask  the  stained  bacteria  as 
to  render  them  difficult,  if  not  impossible,  of  detection. 
Tissues  must,  therefore,  always  be  subjected  to  some 
degree  of  decolorization,  and  this  must  be  accomplished 
without  depriving  the  bacteria  of  their  color. 

The  details  of  the  method  of  decolorization  will  be 
described  in  the  section  on  the  technique  of  staining. 

Another  point  to  be  remembered  in  staining  tissues 
is  that  they  cannot  be  heated  and  retain  their  structure 
in  the  same  way  that  one  heats  cover-slips.  The  best 
results  are  not  obtained  in  efforts  to  hasten  the  staining 
by  subjection  to  high  temperatures,  but  rather  by  longer 
exposures  to  lower  temperatures. 

HARDENING  THE  TISSUES. — The  bits  of  tissue — not 
larger  than  one  cubic  centimetre — are  to  be  placed,  as 
fresh  as  possible,  in  absolute  alcohol.  They  should  rest 
upon  a  pad  of  cotton  or  filter-paper  in  the  bottle  con- 


SECTION-CUTTING.  175 

taining  the  alcohol,  in  order  that  they  may  be  elevated 
and  surrounded  by  the  portion  of  the  alcohol  which  is 
specifically  the  lightest,  and  consequently  contains  least 
water.  The  alcohol  abstracts  water  from  the  tissue, 
and  as  the  dehydration  proceeds  the  tissue  becomes 
accordingly  more  and  more  dense.  When  of  about  the 
consistency  of  fresh  solid  rubber,  or  preferably  not  quite 
so  dense,  it  is  ready  to  cut.  A  small  portion,  about  half 
a  cubic  centimetre,  should  be  cemented  to  a  bit  of  cork 
with  ordinary  mucilage,  and  allowed  to  remain  in  the 
open  air  for  a  minute  or  two  for  the  mucilage  to  harden. 
Alcohol  should  be  dropped  upon  it  occasionally  to  pre- 
vent drying  of  the  tissue.  When  the  mucilage  is  hard 
the  cork  with  the  piece  of  tissue  upon  it  may  be  left  in 
alcohol  over  night,  and  on  the  following  day  the  sec- 
tions may  be  cut. 

SECTION-CUTTING. — This  is  accomplished  by  the  use 
of  an  instrument  known  as  a  microtome.  In  Fig.  36 
is  seen  the  form  now  commonly  employed.  It  is  known 
as  Schanze's  microtome.  It  is  an  apparatus  provided  with 
a  clamp  for  holding  the  cork  upon  which  the  tissue  is  ce- 
mented, and  also  a  sliding  clamp  which  carries  a  knife. 
The  tissue  is  clamped  horizontally,  and  the  knife  is  caused 
to  slide  across  its  upper  surface,  also  in  a  horizontal  plane. 
Beneath  the  clamp  for  holding  the  tissue  is  a  milled  disk, 
by  means  of  which  a  screw  is  caused  to  revolve,  and  in 
revolving  raises  or  lowers  the  clamp  holding  the  tissue, 
so  that  the  tissue  may  be  brought  closer  to  or  farther 
from  the  plane  in  which  the  knife  slides.  By  this 
arrangement  sections  of  any  desired  thickness  can  be 
cut  by  turning  the  milled  disk  with  the  one  hand  and 
causing  the  knife  to  traverse  the  tissue  with  the  other. 
The  tissue  and  the  knife-blade  should  be  kept  wet 


176  BACTERIOLOGY. 

with  alcohol,  so  that  the  sections  may  float  upon  the 
blade  of  the  knife,  from  which  they  can  easily  be  re- 
moved, without  tearing,  with  a  curved  needle  or  a 

FIG.  36. 


Schanze's  microtome. 

camePs-hair  brush.  As  the  sections  are  cut  they  are 
placed  in  a  dish  containing  alcohol. 

There  are  some  tissues  which,  by  reason  of  their 
histological  structure,  do  not  become  sufficiently  dense 
when  exposed  to  alcohol  to  permit  of  their  being  cut  in 
the  above  way.  It  becomes  necessary  to  render  them 
more  solid  by  filling  their  interstices  with  some  sub- 
stance that  neither  interferes  with  their  structure  nor 
prevents  their  being  cut  into  sections.  They  must  be 
"  imbedded/7  as  this  process  is  called. 

Imbedding  in  celloidin.  Most  convenient  for  this 
purpose  is  celloidin,  a  body  somewhat  similar  to  collo- 
dion, soluble  in  a  mixture  of  equal  parts  of  alcohol  and 
ether,  as  well  as  in  absolute  alcohol. 


SECTION-CUTTING.  177 

After  hardening  in  alcohol  the  tissue  to  be  imbedded 
is  placed  in  a  mixture  of  equal  parts  of  absolute  alcohol 
and  ether  and  left  there  for  twenty-four  hours.  It  is 
then  transferred  to  celloidin.  Two  solutions  of  celloidin 
are  to  be  employed  :  the  one  a  thin  solution  in  a  mixture 
of  equal  parts  of  absolute  alcohol  and  ether,  the  other  a 
thick  solution  in  the  same  solvent.  Into  the  thin  solu- 
tion, which  should  be  of  about  the  consistence  of  very 
thin  syrup,  the  tissue  is  placed  from  the  absolute  alcohol 
and  ether,  and  allowed  to  remain  for  twenty-four  hours. 
It  is  then  placed  in  the  thick  solution  for  about  a  day. 
When  removed  it  should  be  placed  immediately  upon 
a  bit  of  cork  or  a  block  of  wood.  The  adherent  cel- 
loidin acts  as  a  cement ;  as  it  hardens  rapidly  the 
tissue  is  soon  fast  to  the  cork.  The  section  is  then 
left  in  60  per  cent,  alcohol  for  twenty-four  hours  to 
complete  the  solidification  of  the  celloidin,  after  which 
sections  may  be  cut  in  the  way  just  described  for  tissues 
not  so  treated. 

Imbedding  in  paraffin.  After  bits  of  the  tissue  not 
larger  than  a  cubic  centimetre  have  been  hardened  in 
the  usual  way  they  are  placed  in  fresh  absolute  alcohol 
for  twenty-four  hours  to  complete  the  process.  From 
thirs  they  are  transferred  to  pure  turpentine,  and  kept  in 
a  warm  oven  at  a  temperature  not  exceeding  35°  to 
38°  C.  Here  they  remain  until  thoroughly  saturated 
with  the  turpentine,  as  is  recognized  by  the  transparent 
appearance  they  assume.  From  this  they  are  placed  in 
paraffin  that  melts  at  53°  C.,  and  allowed  to  remain  in 
this  for  three  or  four  hours.  They  are  then  trans- 
ferred to  a  small  paper  or  metal  mould,  or  a  pill-box, 
and  melted  paraffin  is  poured  over  them.  When  the 
paraffin  has  become  solid  the  mould  or  box  is  removed 
12 


178  BACTERIOLOGY. 

from  around  it,  the  excess  of  paraffin  trimmed  from 
about  the  imbedded  tissue,  and  the  latter  is  ready  for 
cutting. 

When  the  sections  are  cut  they  are  freed  from  par- 
affin by  exposing  them  to  turpentine ;  the  latter  is  re- 
moved by  washing  in  alcohol  and  the  sections  can  then 
be  stained  in  the  ordinary  way.  In  cutting  sections 
from  tissues  that  have  been  imbedded  in  paraffin  the 
long  axis  of  the  knife  should  be  at  nearly  right  angles 
to  the  direction  in  which  the  knife  travels.  For  bacte- 
riological purposes  the  method  of  imbedding  in  paraffin 
does  not,  as  a  rule,  give  such  good  results  as  when  the 
celloidin  method  is  employed.  In  this  work,  therefore, 
the  latter  is  usually  preferred. 

STAINING  THE  SECTIONS. — The  sections  when  cut 
may  be  stained  in  a  variety  of  ways.  The  ordinary 
watery  solutions  of  the  three  common  basic  aniline 
dyes — fuchsin,  gentian-violet,  and  methylene-blue — 
or,  what  is  better,  the  alkaline  methylene-blue  solution 
of  Loffler,  may  be  employed  for  general  use. 

Some  of  the  acid  aniline  dyes,  as  well  as  some  of  the 
vegetable  coloring-matters,  are  essentially  nuclear  stains, 
and  are  not  applicable  to  the  staining  of  bacteria. 

Into  a  watch-glass  containing  either  of  the  staining- 
solutions  mentioned  the  sections  are  to  be  placed  after 
having  been  in  water  for  about  one  minute.  They  re- 
main in  the  staining-solutions  for  from  five  to  eight 
minutes.  They  are  then  removed,  rinsed  in  water,  and 
partly  decolorized  in  0.1  per  cent,  solution  of  acetic  acid 
for  only  a  few  seconds;  again  washed  in  water,  then 
in  absolute  alcohol  for  a  few  seconds,  and  from  this  again 
into  absolute  alcohol  for  the  same  time,  and  finally  into 
cedar-oil  or  xylol.  Here  they  remain  for  from  one-half 


STAINING  THE  SECTIONS.  179 

to  three-fourths  of  a  minute.  They  are  now  to  be  evenly 
spread  upon  a  spatula,  which  is  held  under  them  in 
the  fluid,  and,  without  draining  off  the  fluid,  are 
transferred  to  a  clean  glass  slide.  This  must  be  done 
carefully  to  avoid  tearing.  The  easiest  way  to  do  this 
is  to  hold  the  spatula  on  which  the  section  floats  in  one 
hand,  with  its  point  just  touching  the  surface  of  the 
glass  slide,  and  then  with  a  needle  gently  pull  the 
section  upon  the  slide.  The  fluid  comes  with  it,  and 
the  floating  section  may  be  easily  spread  out  into  a  flat 
surface.  The  excess  of  fluid  is  taken  up  with  blot- 
ting-paper, after  which  a  drop  of  xylol-balsam  is  placed 
upon  the  centre  of  the  section,  which  is  then  covered 
with  a  thin,  clean  cover-slip.  It  is  now  ready  for 
examination. 

Each  step  in  the  above  process  has  its  definite  object. 
The  sections  are  placed  in  water  before  staining  in  order 
that  the  diffusion  of  the  staining-solution  into  the  tis- 
sues may  be  diminished ;  otherwise  our  efforts  at  render- 
ing the  bacteria  more  conspicuous  by  decolorizing  the 
tissues  in  which  they  are  located  would  rob  the  bacteria 
of  their  color  as  well. 

The  acetic  acid  and  also  the  alcohol  are  decolorizers. 
They  are  employed  to  reduce  the  excess  of  stain  in  the 
tissues,  though  they  also  have  some  decolorizing  action 
upon  the  bacteria.  The  cedar-oil  and  xylol  are  bodies 
which  mix  on  the  one  hand  with  alcohol,  and  on  the 
other  with  balsam.  They  are  known  as  "  clearing- 
fluids,"  and  not  only  serve  to  differentiate  the  compo- 
nent parts  of  the  tissue,  but  fill  up  th6  gap  that  would 
otherwise  be  left  in  the  process,  for  a  section  cannot 
be  mounted  in  balsam  directly  from  alcohol ;  the  two 
bodies  do  not  mix  perfectly.  A  number  of  clearing- 


180  BACTERIOLOGY. 

agents  are  in  general  use ;  in  fact,  almost  all  the  essen- 
tial oils  come  under  this  head.  There  is  one — oil  of 
cloves — which  is  very  commonly  used  in  histological 
work ;  but  it  must  not  be  employed  for  tissues  contain- 
ing bacteria.  It  not  only  extracts  too  much  color  from 
the  bacteria,  but  causes  them  to  fade  after  the  sections 
have  been  mounted  for  a  time. 

When  the  section  thus  stained  and  mounted  is  exam- 
ined microscopically  it  may  be  found  that  the  tissues 
still  possess  so  much  color  that  the  bacteria  are  not  vis- 
ible, in  which  case  they  have  not  been  decolorized  suffi- 
ciently ;  or,  on  the  other  hand,  both  bacteria  and  tissues 
may  have  parted  with  their  stains — then  decolorization 
has  been  carried  too  far.  In  either  case  the  fault  must 
be  remedied  in  the  manipulation  of  the  next  section  to 
be  mounted. 

In  short,  the  steps  in  the  process  of  staining  sections 
in  general  are  these  : 

a.  From  alcohol  into  distilled  water  for  one  minute. 

b.  Into  the  stain  ing-fluid  for  from  five  to  eight  min- 
utes. 

c.  Into  water  from  three  to  five  minutes. 

d.  Into  0.1  per  cent,  acetic  acid  for  about  one-half 
minute. 

e.  Into  absolute  alcohol  for  a  few  seconds. 

/.  Into  absolute  alcohol  again  for  a  few  seconds. 

g.  Xylol  for  about  one-half  minute. 

h.  Removal  with  spatula  or  section-lifter  to  slide. 

i.  Removal  of  excess  of  xylol. 

j.  Mounting  in  xylol-balsam. 

The  section  must  be  lifted  from  one  vessel  to  the  other 
by  means  of  either  a  curved  needle  or  a  glass  rod  drawn 
out  to  a  fine  end  and  bent  in  the  form  of  a  curved  needle. 


STAINING  THE  SECTIONS.  181 

By  the  above  process  of  staining,  which  can  be  prac- 
tised as  a  routine  method  for  most  bacteria  in  tissues, 
the  nuclei  of  the  tissue-cells,  as  well  as  the  bacteria,  will 
be  more  or  less  deeply  stained. 

SPECIAL  METHODS  OF  STAINING  BACTERIA  IN 
TISSUES. — For  purposes  of  contrast-stains  it  sometimes 
becomes  necessary  to  decolorize  completely,  or  nearly 
completely,  the  tissues  and  leave  the  bacteria  unaltered 
in  color.  For  this  purpose  special  methods  depending 
on  the  staining-peculiarities  of  the  bacteria  under  con- 
sideration have  been  devised. 

Gram's  method  with  tissues.  One  of  the  most  com- 
monly employed  differential  stains  is  that  of  Gram. 
In  general,  it  is  practised  in  the  wray  given  for  its  em- 
ployment on  cover-slip  preparations,  with  some  slight 
modifications. 

In  this  method  the  sections  are  transferred  from 
water  into  a  solution  of  aniline-water  gentian-violet,  as 
prepared  by  the  Koch-Ehrlich  formula,  but  which  has 
been  diluted  with  about  one-third  its  volume  of  wrater. 
In  this  the  sections  remain  for  about  ten  minutes,  pref- 
erably in  a  warm  place,  at  a  temperature  of  about  40°  C. 
They  should  never,  under  any  conditions,  be  boiled. 
From  this  they  are  washed  alternately  in  the  iodine 
solution  and  alcohol,  occasionally  renewing  the  stained 
with  clean  alcohol,  until  all  color  has  been  extracted 
from  them.  They  are  then  brought  for  one  minute  into 
a  dilute  watery  solution  of  eosin  or  safranin,  or  Bis- 
marck-brown, again  washed  for  a  few  seconds  in 
alcohol,  and  finally  for  one-fourth  minute  in  absolute 
alcohol.  From  this  they  are  transferred  to  xylol  for  a 
half-minute.  The  remaining  steps  in  the  process  are 
the  same  as  those  given  in  the  general  method.  In 


182  BACTERIOLOGY. 

some  cases  better  results  are  obtained  by  reversing  the 
steps  in  the  process  and  staining  the  bacteria  last,  for 
then  the  repeated  decolorizing  action  of  the  alcohol  on 
the  bacteria  is  diminished ;  thus,  place  the  sections  from 
alcohol  into  eosin,  safranin,  or  Bismarck-brown  for  a  few 
minutes,  then  wash  out  in  50  per  cent,  alcohol,  then  for 
from  three  to  five  minutes  in  the  dilute  aniline- water 
gentian-violet  solution,  then  into  the  iodine  bath ;  after 
three  minutes  wash  out  in  alcohol,  and,  finally,  for  one- 
fourth  minute  in  absolute  alcohol,  and  then  into  the 
xylol,  from  which  they  may  be  mounted.  Some  of  the 
organisms  which  may  be  stained  by  this  method  are 
microcoecus  tetragenus,  bacillus  diphtheria?,  bacillus  an- 
thracis,  and  staphylococcus  pyogenes  aureus.  It  cannot 
be  successfully  employed  with  the  bacillus  of  typhoid 
fever,  nor  with  tissues  containing  microcoecus  gonorrhoeas. 

Staining  ivith  dahlia  and  decolorizing  with  sodium  car- 
bonate solution.  Another  method  that  is  not  very  com- 
monly employed,  though  the  results  obtained  by  its  use 
are  in  many  cases  very  satisfactory,  is  to  stain  the  tis- 
sues in  a  strong  watery  solution  of  dahlia  (about  one- 
fourth  saturated)  for  from  ten  to  fifteen  minutes  ;  from 
this  they  are  transferred  to  a  2  per  cent,  solution  of 
sodium  or  potassium  carbonate,  and  from  this  into  alco- 
hol, alternating  from  the  one  to  the  other  until  the 
sections  are  almost  colorless.  After  the  alcohol  they 
are  rinsed  in  water  and  then  put  into  a  dilute  watery 
solution  of  either  eosin,  Bismarck-brown,  or  safranin  for 
one  minute,  then  washed  out  in  alcohol,  finally  in  abso- 
lute alcohol,  and  then  in  xylol,  from  which  they  may  be 
mounted  in  the  manner  given. 

Especially  brilliant  results  are  obtained  when  tissues 
containing  anthrax  bacilli  are  stained  by  this  process ; 


STAINING  THE  SECTIONS.  183 

the  bacilli  will  be  of  a  deep-blue  color,  while  the  sur- 
rounding tissues  will  be  of  the  color  used  as  contrast. 

Kuhnds  carbolic  methylene-blue  method.  Stain  the 
sections  in  the  following  solution  for  from  one-half  to 
one  hour: 

Methylene-blue,  in  substance 1.5  grammes. 

Absolute  alcohol 10    c.c. 

Rub  up  thoroughly  in  a  mortar,  and  when  the  blue 
is  completely  dissolved  add  gradually  100  c.c.  of  a  5 
per  cent,  solution  of  carbolic  acid.  (This  solution  de- 
composes after  a  short  time ;  it  should  be  made  fresh 
when  needed.)  From  this  the  sections  are  washed  in 
water,  then  in  1.5  to  2  per  cent,  hydrochloric  acid  in 
water,  after  which  they  are  transferred  to  a  solution  of 
lithium  carbonate  of  the  strength  of  six  to  eight  drops 
of  a  concentrated  watery  solution  of  the  salt  to  ten  drops 
of  water,  and  from  this  they  are  again  thoroughly 
washed  in  water,  then  in  absolute  alcohol  containing 
enough  methylene-blue  in  substance  to  give  it  a  toler- 
ably dense  color,  then  for  a  few  minutes  in  aniline  oil 
to  which  a  little  methylene-blue  in  substance  has  been 
added,  then  completely  rinsed  out  in  pure  aniline  oil ; 
from  this  they  are  passed  into  thymol  or  oil  of  turpen- 
tine for  two  minutes,  and  then  into  xylol,  from  which 
they  are  mounted  in  xylol-balsam.  The  advantages 
of  this  method  are  that  it  is  generally  applicable,  and 
by  its  use  the  bacteria  are  not  robbed  of  their  color, 
whereas  the  tissues  are  sufficiently  decolorized  to  render 
the  bacteria  visible  and  admit  of  the  use  of  contrast- 
stains. 

Weigert's  modification  of  Gram's  method  for  sections. 
Stain  the  sections  in  the  Koch-Ehrlich  aniline-water 
gentian-violet  solution  for  five  or  six  minutes;  wash 


184  BACTERIOLOGY. 

in  water  or  physiological  salt-solution  (0.6  to  0.7  per 
cent,  solution  of  sodium  chloride  in  distilled  water) ; 
transfer  them  with  the  section-lifter  to  the  slide ;  take 
up  the  excess  of  fluid  by  gently  pressing  upon  the  flat 
section  with  blotting-paper ;  treat  the  section  with  the 
iodine  solution  used  by  Gram ;  take  up  the  excess  of 
the  solution  with  blotting-paper ;  cover  the  section  with 
aniline  oil — this  not  only  differentiates  the  component 
parts  of  the  section,  but  dehydrates  as  well ;  wash  out 
the  aniline  oil  with  xylol,  and  mount  in  the  usual  way 
in  xylol-balsam.  Or,  decolorization  with  iodine  may 
be  omitted,  and  the  sections,  after  staining  in  the  ani- 
line-water gentian-violet  for  five  or  six  minutes — or 
longer,  if  necessary — are  transferred  to  the  slide  without 
being  washed  in  water  or  salt-solution  (or,  if  so,  only 
very  slightly  and  rapidly),  dried  as  completely  as  possi- 
ble with  filter-paper,  and  then  decolorized  with  a  mixt- 
ure of  aniline  oil  (one  part)  and  xylol  (two  parts). 
This  is  the  delicate  part  of  the  process,  and  can  be 
watched  under  a  low  power  of  the  microscope.  When 
decolorization  is  sufficient  (repeated  applications  of  the 
aniline  oil  and  xylol  mixture  are  generally  necessary) 
pure  xylol  replaces  the  mixture,  and  the  specimen  is 
finally  mounted  in  xylol-balsam.  Unless  all  the  ani- 
line oil  is  replaced  by  the  xylol  the  specimen  will  not 
keep  well.  In  this  process  the  aniline  oil  is  really  the 
decolorizer,  and  has  the  valuable  property  of  absorbing 
a  certain  amount  of  water,  so  that  dehydration  with 
alcohol  is  avoided.  This  method,  while  it  stains  certain 
bacteria  in  tissues  very  satisfactorily,  is  nevertheless  de- 
signed especially  for  the  staining  of  fibrin.  Fibrin  and 
hyaline  material  will  be  stained  deep  blue ;  bacteria  a 
dark  violet. 


STAINING  TUBERCLE  BACILLI  IN  TISSUES.   185 

STAINING  TUBERCLE  BACILLI  IN  TISSUES. — As  in 
the  case  of  cover-slips,  only  those  methods  most  com- 
monly employed  will  be  given. 

The  method  of  Ehrlich.  Stain  the  sections  in  aniline- 
water  fuchsin  or  gentian- violet  for  twenty-four  hours ; 
decolorize  in  20  per  cent,  nitric  acid  for  a  few  seconds 
only — the  color  need  not  be  entirely  extracted  ;  then  in 
70  per  cent,  alcohol  until  no  more  color  can  be  extracted 
by  the  alcohol ;  stain  as  contrast-color  in  dilute  watery 
methylene-blue,  malachite-green,  or  Bismarck-brown 
solution  ;  wash  out  in  90  per  cent,  alcohol,  then  in  abso- 
lute alcohol  for  a  few  seconds ;  clear  up  in  xylol  and 
mount  in  xylol-balsam. 

Method  of  Ziehl-Neelsen.  Stain  the  sections  in  warmed 
carbol-fuchsin  solution  for  one  hour ;  temperature  to  be 
about  45°  to  50°  C.  Decolorize  for  a  few  seconds  in  5 
per  cent,  sulphuric  acid,  then  in  70  per  cent,  alcohol, 
and  from  this  on  as  by  the  Ehrlich  method. 

Dry  method.  For  tubercle  bacilli,  as  for  many  other 
organisms  in  tissues,  the  following  method  may  be 
employed  if  only  the  presence  of  organisms  is  to  be 
detected  and  the  histological  condition  of  the  tissues  is 
a  matter  of  no  consequence  :  bring  the  sections  from 
water  upon  a  slide  or  cover-slip,  dry,  fix,  and  stain  by 
the  methods  for  cover-slip  preparations. 

Gray's  method.  The  method  employed  by  Gray  at 
the  Army  Medical  Museum,  Washington,  D.  C.,  a  de- 
scription of  which  is  given  by  Borden,  is  as  follows: 
the  tissue  to  be  stained  should  be  hardened  preferably 
in  alcohol,  in  pieces  not  exceeding  1.5  by  1.5  by  1  cm. 
in  size,  though  tissues  hardened  by  any  other  of  the 
regular  methods  can  be  stained.  Alcohol  is  to  be 
preferred,  however,  as  after  its  use  the  bacilli  stain 


186  BACTERIOLOGY. 

more  quickly  and  brilliantly  than  when  one  of  the 
other  hardening  fluids — Miiller's,  for  instance — is  em- 
ployed. 

After  the  tissue  has  been  hardened  it  is  imbedded  in 
paraffin  and  cut  in  the  usual  manner.  The  sections 
are  then  cemented  to  the  slides  with  a  filtered  ^  per  cent, 
solution  of  gold-label  gelatin,  to  which  is  added  chloral 
hydrate  in  the  proportion  of  1  per  cent.,  as  a  preservative. 
Several  drops  of  this  are  placed  on  each  slide,  a  section 
laid  on  top,  and  the  slides  placed  in  a  warming-oven 
kept  at  a  temperature  slightly  below  the  melting-point 
of  the  paraffin.  In  about  five  minutes  all  wrinkles  will 
have  been  taken  out  of  the  sections,  which  will  lie  per- 
fectly flat  and  smooth  on  the  surface  of  the  gelatin  solu- 
tion. The  slides  are  then  removed  from  the  oven  and 
the  surplus  fluid  poured  from  them,  thus  bringing  the 
sections  in  contact  with  their  surface,  after  which  they 
are  set  aside  in  a  place  protected  from  dust,  to  remain 
until  the  sections  are  firmly  cemented  to  them  by  the 
drying  of  the  gelatin  solution.  The  drying  may  be 
hastened  by  keeping  the  slides  in  an  oven  beloAV  the 
melting-point  of  the  paraffin ;  but  it  is  best  to  set  the 
slides  aside  until  the  next  day,  when  the  sections  will 
be  found  to  be  perfectly  cemented  to  them.  The  par- 
affin is  then  removed  from  the  sections  by  turpentine, 
the  turpentine  by  absolute  alcohol,  the  absolute  alcohol 
by  50  per  cent,  alcohol,  and  this  by  water,  after  which 
the  slides  are  placed  for  five  minutes  in  a  5  per  cent, 
aqueous  solution  of  potassium  bichromate.  This  ren- 
ders the  gelatin  insoluble  and  prevents  the  sections 
leaving  the  slides  during  their  necessarily  more  or  less 
prolonged  immersion  in  the  fuchsin  stain.  The  potas- 
sium bichromate  is  washed  out  with  water,  and  the  slides 


STAINING   TUBERCLE  BACILLI  IN  TISSUES.   187 

are  then  placed  in  a  fuchsin  stain,  which  is  prepared  as 
follows : 

Fuchsin 1.5  grammes. 

Absolute  alcohol 14    c.c. 

Carbolic  acid  crystals  (pure) 6    grammes. 

Water 100    c.c. 

Dissolve  the  fuchsin  in  the  alcohol  and  the  carbolic 
acid  in  the  water.  Mix  the  two  solutions  and  let  stand 
for  twelve  hours,  with  occasional  shaking  or  stirring ; 
then  filter. 

The  length  of  time  that  the  slide  remains  in  this  solu- 
tion varies  with  circumstances.  Tubercle  bacilli  stain 
very  quickly ;  in  tissues  properly  hardened  in  alcohol 
five  minutes  are  generally  sufficient  to  stain  them  deeply. 

Prolonged  immersion  in  the  fuchsin  does  no  harm 
and  insures  certainty  of  results.  After  a  section  has 
been  in  the  stain  a  sufficient  length  of  time  it,  with  the 
slide  to  which  it  is  cemented,  is  washed  in  water  until 
the  surplus  stain  is  removed ;  it  is  then  subjected  to  the 
action  of  a  combined  decolorizer  and  contrast-stain  made 
as  follows : 

Methyl-blue .  2.25  grammes 

Absolute  alcohol .   ,  30      c.c. 

Sulphuric  acid '•....  12 

Water  (distilled) 100 

Dissolve  the  methyl-blue  in  the  alcohol,  add  the  acid 
to  the  water,  mix  the  two  solutions,  and  let  stand,  with 
occasional  shaking,  for  twelve  hours ;  then  filter. 

This  solution  is  allowed  to  act  upon  the  tissue  for  a 
few  seconds,  and  as  soon  as  the  blue  color  predominates 
over  the  red,  as  seen  by  transmitted  light,  the  section  is 
immediately  washed  in  water.  Generally  the  red  color 


188  BACTERIOLOGY. 

reappears,  and  the  section  must  be  again  subjected  to 
the  action  of  the  blue  solution  and  again  washed  in 
water.  This  must  be  repeated  until  the  blue  almost, 
if  not  quite  completely  and  permanently,  replaces  the 
red  stain.  This  is  the  most  important  part  of  the  proc- 
ess, and  entirely  satisfactory  results  are  only  obtained 
after  some  practice.  The  tendency  is  usually  not  to 
replace  sufficiently  the  fuchsin  with  the  methyl-blue, 
and  in  consequence  the  red  color  of  the  bacilli  is  masked 
by  the  red  of  the  surrounding  tissues.  Unless  all  acid 
is  thoroughly  removed  by  the  final  washing  in  water 
the  stain  is  not  permanent.  The  section  is  then  com- 
pletely dehydrated  with  absolute  alcohol,  after  taking 
up  the  excess  of  water  on  the  slide  with  blotting-paper. 
The  alcohol  is  followed  by  turpentine,  and  the  process 
is  completed  by  mounting  in  xylol-balsam. 

In  case  it  is  desired  to  stain  sections  cut  by  the  freez- 
ing method,  they  are  placed  upon  a  slide  on  which  a 
few  drops  of  the  gelatin  fixative  have  been  placed,  and 
after  about  five  minutes,  during  which  the  fixative  will 
have  penetrated  the  section,  the  surplus  is  poured  from 
beneath  the  section.  The  slides  are  then  set  aside  for 
the  gelatin  to  harden  by  drying,  and  after  drying  they 
are  placed  in  bichromate  fluid  to  render  the  gelatin 
insoluble.  They  are  then  manipulated  in  exactly  the 
same  manner  as  the  sections  cut  by  the  paraffin  method. 

This  method  gives  equally  as  good  results  with  tissues 
containing  the  lepra  bacillus  as  with  those  containing 
tubercle  bacilli. 


CHAPTER  XI. 

Systematic  study  of  an  organism — Points  to  be  considered  in  identi- 
fying an  organism  as  a  definite  species. 

AFTER  isolating  an  organism  by  the  plate  method 
considerable  work  is  necessary  in  order  to  establish  its 
identity  as  a  definite  species.  It  must  possess  certain 
morphological  and  cultural  peculiarities,  which  must  be 
constant  under  constant  conditions.  Its  form  at  dif- 
ferent stages  must  always  be  the  same.  Its  ability  or 
inability  to  produce  spores  must  not  vary  under  proper 
conditions.  Its  growth  upon  different  media  under 
constant  conditions  of  temperature  and  reaction  must 
always  present  the  same  outward  appearances.  The 
changes  brought  about  by  it  in  the  reaction  of  the 
media  in  which  it  is  growing  must  follow  a  fixed  rule. 
Its  power  to  produce  liquefaction  of  the  gelatin,  or  to 
grow  upon  it  without  bringing  about  this  change,  must 
always  be  the  same.  Its  motility  or  non-motility, 
and,  if  motile,  the  approximate  number  and  position  of 
its  organs  of  locomotion,  must  be  determined.  Its  pro- 
duction of  certain  chemical  products  must  be  established 
by  chemical  analysis.  Its  behavior  toward  oxygen — 
/.  e.,  Does  it  require  this  gas  for  its  growth  ?  Is  this  gas 
an  indifferent  factor  ?  or,  By  its  presence  are  the  life- 
processes  of  the  organism  checked  ? — must  be  decided. 
Its  behavior  under  varying  conditions  of  temperature 
and  under  the  influence  of  different  chemical  bodies,  as 
well  as  its  growth  in  media  of  different  reactions,  is 

189 


190  BACTERIOLOGY. 

to  be  studied.  The  property  of  producing  fermentation 
with  the  liberation  of  gases,  and  the  character  and  quan- 
titative relations  of  these  gases,  must  be  ascertained.  If 
it  produces  pigment,  what  are  the  conditions  favorable 
and  unfavorable  to  this  function.  And,  lastly,  we  must 
consider  its  behavior  when  introduced  into  the  bodies 
of  animals  used  for  experimental  work — i.  e.,  Is  it  a 
disease-producing  organism,  or  does  it  belong  to  the 
group  of  innocent  saprophytes? 

We  have  learned  the  methods  of  obtaining  colonies, 
and  have  acquainted  ourselves  with  some  of  the  pecu- 
liarities by  which  they  are  distinguished  from  one 
another.  The  next  important  steps  are  to  determine  the 
morphology  of  the  individuals  composing  these  colonies, 
as  well  as  their  relation  to  each  other  in  the  colony. 
These  points  are  decided  by  microscopic  examination  of 
bits  of  the  colony,  which  arc  transferred  to  thin  glass 
cover-slips,  upon  which  they  are  dried,  stained,  and 
mounted.  Cover-slips  for  this  purpose  are  prepared  in 
two  ways :  either  by  taking  up  a  bit  of  the  colony  on  a 
platinum  needle,  smearing  it  upon  a  cover-slip,  staining 
it,  and  examining  it — by  which  only  the  morphology  of 
the  individual  bacteria  can  be  made  out — or  by  the 
method  of  "  impression  cover-slip  preparations,"  by 
which  not  only  the  morphology,  but  also  the  relation  of 
the  organisms  in  the  colony  to  one  another  can  be  deter- 
mined. The  details  of  these  methods  will  be  found  in 
the  chapter  on  the  Methods  of  Staining. 

MICROSCOPIC   EXAMINATION   OF   PREPARATIONS. 

THE  DIFFERENT  PARTS  OF  THE  MICROSCOPE. — 
Before  describing  the  method  of  examining  prepara- 
tions microscopically,  a  few  definitions  of  the  terms 


DIFFERENT  PARTS  OF   THE  MICROSCOPE.    191 


used  in  referring  to  the  microscope  may  not  be  out  of 
place.  (The  different  parts  of  the  microscope  referred 
to  below  are  indicated  by  letters  in  Fig.  37.) 


FIG.  37. 


The  ocular  or  ei/e-piec-e  (A)  is  the  lens  at  which  the 
eye  is  placed  when  looking  through  the  instrument.     It 


192  BACTERIOLOGY. 

serves  to  magnify  the  image  projected  through  the  ob- 
jective. 

The  objeetive  (B)  is  the  lens  which  is  at  the  distal 
end  of  the  barrel  of  the  instrument,  and  which  serves 
to  magnify  the  object  to  be  examined. 

The  stage  (c)  is  the  shelf  or  platform  of  the  micro- 
scope on  which  the  object  to  be  examined  rests. 

The  diaphragms  are  the  perforated  stops  that  fit  in 
the  centre  of  the  stage.  They  vary  in  size,  so  that  dif- 
ferent amounts  of  light  may  be  admitted  to  the  object 
by  using  diaphragms  with  larger  or  smaller  openings. 

The  "  iris "  diaphragm  (D)  opens  and  closes  like  the 
iris  of  the  eye.  It  is  so  arranged  that  its  opening  for 
admission  of  light  can  be  increased  or  diminished  by 
moving  a  small  lever  in  one  or  another  direction. 

The  reflector  (E)  is  the  mirror  placed  beneath  the 
stage,  which  serves  to  illuminate  the  object  to  be  exam- 
ined. 

The  coarse  adjustment  (F)  is  the  rack-and-pinion  ar- 
rangement by  which  the  barrel  of  the  microscope  can 
be  quickly  raised  or  lowered. 

The  fine  adjustment  (G)  serves  to  raise  and  lower  the 
barrel  of  the  instrument  very  slowly  and  gradually. 

For  the  microscopic  study  of  bacteria  it  is  essential 
that  the  microscope  be  provided  with  an  oil-immersion 
system  and  a  sub-stage  condensing  apparatus. 

The  oil-immersion  or  homogeneous  system  consists  of 
an  objective  so  constructed  that  it  can  only  be  used  when 
the  transparent  media  through  which  the  light  passes  in 
entering  it  are  all  of  the  same  index  of  refraction — i.  e., 
are  homogeneous.  This  is  accomplished  by  interposing 
between  the  face  of  the  lens  and  the  cover-slip  covering 
the  object  to  be  examined  a  body  which  refracts  the 


EXAMINATION  OF  COVER-SLIPS.  193 

light  in  the  same  way  as  do  the  glass  slide,  the  cover- 
slip,  and  the  glass  of  which  the  objective  is  made.  For 
this  purpose  a  drop  of  oil  of  the  same  index  of  refrac- 
tion as  the  glass  is  placed  upon  the  face  of  the  lens, 
and  the  examinations  are  made  through  this  oil.  There 
is  thus  no  loss  of  light  from  deflection,  as  is  the  case  in 
the  dry  system. 

The  sub-stage  condensing  apparatus  (H)  is  a  system 
of  lenses  situated  beneath  the  central  opening  of  the 
stage.  They  serve  to  condense  the  light  passing  from 
the  reflector  to  the  object  in  such  a  way  that  it  is 
focussed  upon  the  object,  thus  furnishing  the  greatest 
amount  of  illumination.  Between  the  condenser  and 
reflector  is  placed  the  "  iris "  diaphragm,  the  aperture 
of  which  can  be  regulated,  as  circumstances  require,  to 
permit  of  either  a  very  small  or  a  very  large  amount  of 
light  passing  to  the  object. 

The  nose-piece  (i)  consists  of  a  collar,  or  group  of 
collars  joined  together  (two  or  more),  that  is  attached  to 
the  distal  end  of  the  tube  of  the  microscope.  It  enables 
one  to  attach  several  objectives  to  the  instrument  in 
such  a  way  that  by  simply  rotating  the  nose-piece  the 
various  lenses  of  different  power  may  be  conveniently 
used  in  succession. 

MICROSCOPIC  EXAMINATION  OF  COVER-SLIPS. — The 
stained  cover-slip  is  to  be  examined  with  the  oil-immer- 
sion objective,  and  with  the  diaphragm  of  the  sub-stage 
condensing  apparatus  open  to  its  full  extent.  The  object 
trained  by  allowing  the  light  to  enter  in  such  a  large  vol- 
ume is  that  the  contrast  produced  by  the  colored  bacteria 
in  the  brightly  illuminated  field  is  much  more  conspicu- 
ous than  when  a  smaller  amount  of  light  is  thrown  upon 
them.  This  is  true  not  only  for  stained  bacteria  on 

13 


194  BACTERIOLOGY, 

cover-slips,  but  likewise  for  their  differentiation  from 
surrounding  objects  when  they  are  located  in  tissues. 
With  unstained  bacteria  and  tissues,  on  the  contrary,  the 
structure  can  best  be  made  out  by  reducing  the  bundle 
of  light-rays  to  the  smallest  amount  compatible  with 
distinct  vision,  and  in  this  way  favoring,  not  color-con- 
trast, but  contrasts  which  appear  as  lights  and  shadows, 
due  to  the  differences  in  permeability  to  light  of  the 
various  parts  of  the  material  under  examination. 

STEPS  IN  EXAMINING  STAINED  PREPARATIONS 
WITH  THE  OIL-IMMERSION  SYSTEM. — Place  upon  the 
centre  of  the  cover-slip  which  covers  the  preparation  a 
small  drop  of  immersion  oil.  Place  the  slide  upon  the 
centre  of  the  stage  of  the  microscope.  With  the  coarse 
adjustment  lower  the  oil-immersion  objective  until  it 
just  touches  the  drop  of  oil.  Open  the  illuminating 
apparatus  to  its  full  extent.  Then,  with  the  eye  to  the 
ocular  and  the  hand  on  the  fine  adjustment,  turn  the 
adjusting-screw  toward  the  right  until  the  field  becomes 
somewhat  colored  in  appearance.  When  this  is  seen 
proceed  more  slowly  in  the  same  direction,  and,  after 
one  or  two  turns,  the  object  will  be  in  focus.  Do  not 
remove  the  eye  from  the  instrument  until  this  has  been 
accomplished. 

Then,  with  one  hand  upon  the  fine  adjustment  and 
the  thumb  and  index  finger  of  the  other  hand  hold- 
ing the  slide  lightly  by  its  end,  it  may  be  moved  about 
under  the  objective.  At  the  same  time  the  screw 
of  the  fine  adjustment  must  be  turned  back  and  forth, 
so  that  the  different  planes  of  the  preparation  may  be 
brought  into  focus  one  after  the  other.  In  this  way  the 
whole  section  or  preparation  may  be  inspected.  When 
the  examination  is  finished  raise  the  objective  from  the 


UNSTAINED  PREPARATIONS.  195 

preparation  by  turning  the  screw  of  the  coarse  adjust- 
ment toward  you.  Remove  the  preparation  from  the 
stage,  and,  with  a  fine  silk  cloth  or  handkerchief,  wipe 
very  gently  and  carefully  the  oil  from  the  face  of  the  lens. 
The  lens  is  then  unscrewed  from  the  microscope  and 
placed  in  the  case  intended  for  its  reception. 

During  work,  of  course,  the  lens  need  not  be  cleaned 
and  put  away  after  each  examination ;  but  when  the 
work  for  the  day  is  over  an  immersion  lens  must 
always  be  protected  in  this  way.  Under  no  circum- 
stances should  it  be  allowed  to  remain  in  the  immersion 
oil  or  exposed  to  dust  for  any  length  of  time. 

EXAMINATION  OF  UNSTAINED  PREPARATIONS. — 
"  Hanging  drops."  It  frequently  becomes  necessary  to 
examine  bacteria  in  the  unstained  condition.  The  cir- 
cumstances calling  for  this  arise  while  studying  the 
multiplication  of  cells,  the  germination  of  spores,  the 
formation  of  spores,  and  the  absence  or  presence  of 
motility. 

In  this  method  the  organisms  to  be  studied  are  sus- 
pended in  a  drop  of  physiological  salt-solution  or  bou- 
illon in  the  centre  of  a  cover-slip.  This  is  then  placed, 
drop  down,  upon  a  slide  in  the  centre  of  which  a  hollow 
or  depression  is  ground  (Fig.  38).  The  slip  is  held  in 

FIG.  38. 


Longitudinal  section  of  hollow-ground  glass  slide  for  observing  bacteria  in 
hanging  drops. 

position  by  a  thin  layer  of  vaselin,  which  may  be 
painted  around  the  margins  of  the  depression.  This 
not  only  prevents  the  slip  changing  its  position  during 
examination,  but  also  prevents  drying  by  evaporation 


196  BACTERIOLOGY. 

if  the  preparation  is  to  be  observed  for  any  length 
of  time.  This  is  known  as  the  "  hanging-drop " 
method  of  examination  or  cultivation.  It  is  indispen- 
sable for  the  purposes  mentioned,  and  at  the  same  time 
requires  considerable  care  in  its  manipulation.  The 
fluid  is  so  transparent  that  the  cover-slip  is  often  broken 
by  the  objective  being  brought  down  upon  the  prepara- 
tion before  one  is  aware  that  the  focal  distance  has  been 
reached.  This  may  be  avoided  by  grasping  the  slide 
with  the  left  hand  and  moving  it  back  and  forth  under 
the  objective  as  it  is  moved  toward  the  object.  As  soon 
as  the  least  pressure  is  felt  upon  the  slide  the  objective 
must  be  raised,  otherwise  the  cover-slip  will  be  broken 
and  the  lens  may  be  rendered  worthless. 

A  safer  plan  is  to  bring  the  edge  of  the  drop  into  the 
centre  of  the  field  with  one  of  the  higher  power  dry 
lenses.  When  this  is  accomplished  substitute  the  im- 
mersion for  the  dry  system,  when  the  edge  of  the  drop 
is  readily  detected  with  the  higher  power  lens  some- 
where near  the  centre  of  the  field. 

In  examining  bacteria  by  this  method  there  is  a  pos- 
sibility of  error  that  must  be  guarded  against.  All 
microscopic  insoluble  particles  in  suspension  in  fluids 
possess  a  peculiar  tremor  or  vibratory  motion,  the  so- 
called  "  Brownian  motion."  This  is  very  apt  to  give 
the  impression  that  the  organisms  under  examination 
are  motile,  when  in  truth  they  are  not  so,  their  move- 
ment in  the  fluid  being  only  this  molecular  tremor. 

The  difference  between  the  motion  of  bodies  under- 
going this  molecular  tremor  and  that  possessed  by  cer- 
tain living  bacteria  is  that  the  former  particles  never 
move  from  their  place  in  the  field,  while  living  bac- 
teria alter  their  position  in  relation  to  the  surround- 


STUDY  OF  SPORE-FORMATION.  197 

ing  organisms,  and  may  dart  from  one  position  in  the 
field  to  another.  In  some  cases  the  true  movement  of 
bacteria  is  very  slow  and  undulating,  while  in  others  it 
is  rapid  and  darting.  The  molecular  tremor  may  be 
seen  with  non-motile  and  with  dead  organisms. 

NOTE. — Prepare  three  hanging-drop  preparations — 
one  from  a  drop  of  dilute  India-ink,  a  second  from  a 
culture  of  micrococci,  and  a  third  from  a  culture  of  the 
bacillus  of  typhoid  fever.  In  what  way  do  they  differ  ? 

STUDY  OF  SPORE-FORMATION. — The  hanging-drop 
method  just  mentioned  is  not  only  employed  for  detect- 
ing the  motility  of  an  organism,  but  also  for  the  study 
of  its  mode  of  spore-formation. 

Since  with  aerobic  organisms  spore-formation  occurs, 
as  a  rule,  only  in  the  presence  of  oxygen,  and  is  induced 
more  by  limitation  of  the  nutrition  of  the  organisms 
than  by  any  other  factor,  it  is  essential  that  these  two 
points  should  be  borne  in  mind  in  preparing  the  drop- 
cultures  in  which  the  process  is  to  be  studied.  For  this 
reason  the  drop  of  bouillon  should  be  small  and  the 
air-chamber  relatively  large. 

The  cover-slip  and  hollow-ground  slide  should  be 
carefully  sterilized,  and  with  a  sterilized  platinum  loop 
a  very  small  drop  of  bouillon  is  placed  in  the  centre 
of  the  cover-slip.  The  slip  is  then  inverted  over 
the  hollow  depression  in  the  sterilized  object-glass  and 
sealed  with  vaselin.  The  most  convenient  method  of 
performing  this  last  step  in  the  process  is  to  paint  a 
ring  of  vaselin  around  the  edges  of  the  hollow  in  the 
slide,  and  then,  without  taking  the  cover-slip  from  the 
table  upon  which  it  rests,  invert  the  hollow  over  the 


198  BACTERIOLOGY. 

drop  and  press  it  gently  down  upon  the  cover-slip.  The 
vaselin  causes  the  slip  to  adhere  to  the  slide,  so  that  it 
can  be  easily  taken  up.  The  drop  now  hangs  in  the 
centre  of  the  small  air-tight  chamber  which  exists  be- 
tween the  depression  in  the  slide  and  the  cover-slip. 
(See  Fig.  38.) 

A  very  thin  drop  of  sterilized  agar-agar  may  be  sub- 
stituted for  the  bouillon.  It  serves  to  retain  the  organ- 
isms in  a  fixed  position,  and  the  process  may  be  more 
easily  followed. 

As  soon  as  finished  the  preparation  is  to  be  examined 
microscopically  and  the  condition  of  the  organisms 
noted.  It  is  then  to  be  retained  in  a  warm  chamber 
especially  devised  for  the  purpose,  and  kept  under  con- 
tinuous observation.  The  form  of  chamber  best  adapted 
to  the  purpose  is  one  which  envelops  the  whole  micro- 
scope. It  is  provided  with  a  window  through  which 
the  light  enters,  and  an  arrangement  by  which  the  slide 
may  be  moved  from  the  outside.  The  formation  of 
spores  requires  a  much  longer  time  than  the  germina- 
tion of  spores  into  bacilli,  but  with  patience  both  proc- 
esses may  be  satisfactorily  observed. 

It  will  be  noticed  that  the  description  of  this  process 
is  very  much  like  that  which  immediately  precedes,  but 
differs  from  it  in  one  respect,  viz.,  that  in  this  manipu- 
lation we  are  not  making  a  preparation  which  is  simply 
to  be  examined  and  then  thrown  aside,  but  it  is  an 
actual  pure  culture,  and  must  be  kept  as  such,  otherwise 
the  observation  will  be  worthless.  For  this  reason  the 
greatest  care  must  be  observed  in  the  sterilization  of 
all  objects  employed.  Studies  upon  spore-formation  by 
this  method  frequently  continue  over  hours,  and  some- 
times days,  and  contamination  must,  therefore,  be  care- 


STUDY  OF  GELATIN  CULTURES.  199 

fully  guarded  against.  The  study  should  be  begun  with 
the  vegetative  form  of  the  organisms ;  the  hanging-drop 
preparation  should,  for  this  reason,  always  be  made 
from  a  perfectly  fresh  culture  of  the  organism  under 
consideration  before  time  has  elapsed  for  spores  to  form. 

The  simple  detection  of  the  presence  or  absence  of 
spore-formation  can  in  many  cases  be  made  by  other 
methods.  For  example,  many  species  of  bacteria  which 
possess  this  property  form  spores  most  readily  upon 
media  from  which  it  is  somewhat  difficult  for  them  to 
obtain  the  necessary  nourishment ;  potatoes  and  agar-agar 
that  have  become  a  little  dry  offer  very  favorable  con- 
ditions, because  of  the  limited  area  from  which  the 
growing  bacteria  can  draw  their  nutritive  supplies,  and 
because  of  the  free  access  which  they  have  to  oxygen, 
for,  their  growth  being  on  the  surface,  they  are  sur- 
rounded by  this  gas  unless  means  are  taken  to  prevent 
it.  By  the  hanging-drop  method,  however,  more  than 
this  specific  property  may  be  determined.  It  is  possible 
not  only  to  detect  the  stages  and  steps  in  the  formation 
of  endogenous  spores,  but  when  the  spores  are  com- 
pletely formed  their  germination  into  mature*  rods  may 
be  seen  by  transferring  them  to  a  fresh  bouillon-drop 
or  drop  of  agar-agar  preserved  in  the  same  way.  The 
word  rods  is  used  because  we  have  as  yet  no  evidence 
that  endogenous  spore-formation  occurs  in  any  of  the 
other  morphological  groups  of  bacteria. 

STUDY  OF  GELATIN  CULTURES. — As  has  been  pre- 
viously stated,  the  behavior  of  bacteria  toward  gelatin 
differs — some  of  them  producing  apparently  no  altera- 
tion in  the  medium,  while  the  growth  of  others  is 
accompanied  by  an  enzymotic  action  that  results  in 
liquefaction  of  the  gelatin  at  and  around  the  place  at 


200  BACTERIOLOGY. 

which  the  colonies  are  growing.  In  some  instances 
this  liquefaction  spreads  laterally  and  downward,  caus- 
ing a  saucer-shaped  excavation  ;  while  in  others  the 
colony  sinks  almost  vertically  into  the  gelatin  and  may 
be  seen  lying  at  the  bottom  of  a  funnel-shaped  depres- 
sion. These  differences  are  constantly  employed  as 
one  of  the  means  of  differentiating  otherwise  closely 
allied  members  of  the  same  family  of  bacteria.  (See 
Fig.  34.)  Studies  upon  the  spirillum  of  Asiatic 
cholera  and  a  number  of  closely  allied  species,  for 
example,  reveal  decided  differences  in  the  form  of 
liquefaction  produced  by  these  various  organisms. 
The  minutest  detail  in  this  respect  must  be  noted,  and 
its  frequency  or  constancy  under  varying  conditions 
determined. 

CULTURES  ON  POTATO. — A  very  important  feature 
in  the  study  of  an  organism  is  its  growth  on  sterilized 
potato.  Many  organisms  present  appearances  under 
this  method  of  cultivation  which  alone  can  almost  be 
considered  characteristic.  In  some  cases  coarsely  lob- 
ulated,  elevated,  dry  or  moist  patches  of  development 
occur  after  a  few  hours ;  again,  the  growth  may  be  finely 
granular  and  but  slightly  elevated  above  the  surface  of 
the  potato ;  at  one  time  it  will  be  dry  and  dull  in  ap- 
pearance, again  it  may  be  moist  and  glistening.  Some- 
times bubbles,  due  to  the  fermentative  action  of  the 
growing  bacteria  on  the  carbohydrates  of  the  potato, 
are  produced. 

A  most  striking  form  of  development  on  potato  is  that 
often  exhibited  by  the  bacillus  of  typhoid  fever  and  the 
bacillus  of  diphtheria.  After  inoculation  of  a  potato 
with  either  of  these  organisms  there  is  usually  no  naked- 
eye  evidence  of  growth,  though  microscopic  examina- 


REACTIONS  PRODUCED  BY  BACTERIA.        201 

tion  of  scrapings  from  the  surface  of  the  potato  reveals 
an  active  multiplication  of  the  organisms  which  had 
been  planted  there.  The  potato  is  one  of  the  most 
important  differential  media  that  we  possess  for  this 
work. 

REACTIONS  PRODUCED  BY  BACTERIA  DURING  THEIR 
GROWTH. — The  reactions  produced  in  media  by  dif- 
erent  species  of  bacteria  in  the  course  of  their 
growth  are  very  valuable  as  means  of  differentiation. 
In  some  cases  these  changes  are  so  marked  that  they 
are  readily  detected  by  the  coarser  reagents ;  again,  they 
are  so  slight  as  to  require  the  employment  of  the  most 
delicate  indicators.  Bacteria  sometimes  produce  at  one 
period  of  their  growth  an  alkaline,  at  another  period 
an  acid  reaction.  This  is  observed  in  cultures  of  the 
bacillus  diphtherwe  of  Loffler,  when  the  media  contain 
traces  of  fermentable  carbohydrates. 

These  differences  are  best  seen  after  the  addition  to 
the  media  in  which  the  organisms  are  to  grow  of  one  of 
the  chemical  substances  which  do  not  interfere  with  the 
development  of  the  organisms,  but  of  which  the  color 
varies  with  the  reaction,  the  change  of  reaction  being  in- 
dicated by  the  play  of  color.  Such  substances  as  litmus, 
in  the  form  of  the  so-called  "  litmus  tincture,"  and  co- 
ralline (rosolic  acid)  in  alcoholic  solution,  are  commonly 
employed  for  this  purpose.  They  may  be  added  to  the 
media  in  the  proportions  given  in  the  chapter  on  media, 
and  the  changes  in  their  colors  studied  with  different 
bacteria.  Milk  and  litmus  tincture  or  peptone  solution 
to  which  rosolic  acid  has  been  added  are  excellent  media 
for  this  experiment. 

In  milk  coagula  now  and  then  appear  as  a  result  of 
the  influence  of  acids,  produced  from  milk-sugar  by 


202  BACTERIOLOGY. 

bacterial  action,  upon  the  casein  of  the  milk,  while 
at  times  acids  may  be  produced  and  yet  no  coagula- 
tion take  place. 

ANILINE  DYES  FOR  DIFFERENTIAL  DIAGNOSIS. — 
The  addition  to  solid  media  of  some  of  the  aniline 
dyes,  fuchsin,  methylene-blue,  methylene-green,  and 
several  others,  as  well  as  combinations  of  these  dyes, 
has  been  recommended  as  a  means  of  differentiation 
of  bacteria.  The  changes  that  are  said  to  occur  as  a 
result  of  bacterial  growth  in  media  so  treated  consist 
of  alterations  in  the  color  of  the  media  due  to  the 
oxidizing  or  reducing  properties  of  the  growing  bac- 
teria. As  yet,  but  little  advantage  has  been  derived 
from  this  method  of  work.  It  cannot  at  present  be 
recommended  as  a  reliable  means  of  diagnosis. 

BEHAVIOR  TOWARD  STAINING-REAGENTS. — The  be- 
havior of  certain  bacteria  toward  the  different  dyes 
and  their  reactions  under  special  methods  of  after- 
treatment  aid  materially  in  their  identification.  With 
very  few  exceptions  bacteria  stain  readily  with  the  com- 
mon aniline  dyes ;  but  they  differ  markedly  in  the  te- 
nacity with  which  they  retain  these  colors  under  the 
subsequent  treatment  with  decolorizing-agents. 

The  tubercle  bacillus  and  the  bacillus  of  leprosy,  for 
example,  are  difficult  to  stain  ;  but  when  once  stained 
they  retain  their  color  under  the  action  of  such  energetic 
decolorizing-agents  as  alcohol,  nitric  acid,  oxalic  acid, 
etc.  Certain  other  organisms  when  stained  with  a  solu- 
tion of  gentian-violet  in  aniline-water  retain  their  color 
when  treated  with  such  decolorizing-bodies  as  iodine 
solution  and  alcohol  (Gram's  method),  while  again 
others  are  completely  decolorized  by  this  method. 
Many  of  them  can  only  be  washed  in  water,  or  but 


FERMENTATION.  203 

for  a  few  seconds  in  alcohol,  without  losing  their 
color.  It  is  essential  that  all  these  peculiarities  should 
be  carefully  noted  in  studying  an  organism. 

FERMENTATION. — The  production  of  gas  as  an  indi- 
cation of  fermentation  is  an  accompaniment  of  the 
growth  of  certain  bacteria.  This  is  best  studied  in 
media  to  which  1  to  2  per  cent,  of  grape-sugar  (glucose) 
has  been  added.  In  this  experiment  the  test-tube 
should  be  filled  to  about  one-half  with  agar-agar.  The 
medium  is  then  liquefied,  and  when  at  the  proper  tem- 
perature a  small  quantity  of  a  pure  culture  of  the  organ- 
ism under  consideration  should  be  carefully  distributed 
through  it.  The  tube  is  then  placed  in  ice- water  and 
rapidly  solidified  in  the  vertical  position.  When  solid 
it  is  placed  in  the  incubator.  After  twenty-four  to 
thirty-six  hours,  if  the  organism  possesses  the  property 
of  causing  fermentation  of  glucose,  the  medium  will 
be  dotted  everywhere  with  very  small  cavities  contain- 
ing the  gas  that  has  resulted. 

This  property  of  fermentation  with  evolution  of  gas 
is  of  such  importance  as  a  differential  characteristic  that 
latterly  considerable  attention  has  been  given  to  it,  and 
those  who  have  been  most  intimately  concerned  in  the 
development  of  our  knowledge  on  the  subject  do  not 
consider  it  sufficient  to  say  that  the  growth  of  an  organ- 
ism "is  accompanied  by  the  production  of  gas-bub- 
bles," but  that  under  given  conditions  we  should  deter- 
mine not  only  the  amount  of  gas  or  gases  produced 
by  the  organism  under  consideration,  but  also  their 
nature.  For  this  purpose  Smith1  recommends  the 

1  An  excellent  and  exhaustive  contribution  to  this  subject  has  been 
made  by  Theobald  Smith  in  the  Wilder  Quarter-Century  Book,  Ithaca, 
N.  Y.,  1893. 


204  BACTERIOLOGY. 

employment  of  the  fermentation-tube.  This  is  a  tube 
bent  at  an  acute  angle,  closed  at  one  end  and  enlarged 
with  a  bulb  at  the  other.  At  the  bend  the  tube  is 
constricted.  To  it  a  glass  foot  is  attached  so  that 
the  tube  may  stand  upright.  (See  Fig.  39.)  To  fill 
the  tube  the  fluid  (it  is  only  used  with  fluid  media) 

FIG.  39. 


Fermentation-tube. 

is  poured  into  the  bulb  until  this  is  about  half  full. 
The  tube  is  then  tilted  until  the  closed  arm  is  nearly 
horizontal,  so  that  the  air  may  flow  out  into  the  bulb 
and  the  fluid  flow  into  the  closed  arm  to  take  its 
place.  When  this  has  been  completely  filled  enough 
fluid  should  be  added  to  cover  the  lowest  expanding 
portion  of  the  bulb,  and  the  opening  of  the  bulb  plugged 
with  cotton.  The  tubes  thus  filled  are  then  to  be  ster- 
ilized. During  sterilization  they  are  to  be  maintained 
in  the  upright  position.  Under  the  influence  of  heat  the 


FERMENTATION.  205 

tension  of  the  water- vapor  in  the  closed  arm  forces  most 
of  the  fluid  into  the  bulb.  As  the  tube  cools  the  fluid 
returns  to  its  place  in  the  closed  arm  and  fills  it  again, 
with  the  exception  of  a  small  space  at  the  top,  which  is 
occupied  by  the  air  originally  dissolved  in  the  liquid 
and  which  has  been  driven  out  by  the  heat.  The  air- 
bubble  should  be  tilted  out  after  each  sterilization ;  and 
finally,  after  the  third  exposure  to  steam,  this  arm  of 
the  tube  will  be  free  from  air.  The  medium  employed 
is  bouillon  containing  some  fermentable  carbohydrate, 
as  glucose,  lactose,  or  saccharose.  After  inoculation  the 
flasks  are  placed  in  the  incubator,  and  the  amount  of 
gas  that  collects  in  the  closed  arm  is  noted  from  day  to 
day.  From  studies  that  have  been  made  this  gas  is 
found  to  consist  usually  of  about  one  part  by  volume  of 
carbonic  acid  and  two  parts  by  volume  of  an  explosive 
gas  consisting  largely  of  hydrogen.  For  determining  the 
nature  and  quantitative  relations  of  these  gases  Smith l 
recommends  the  following  procedure  :  "  The  bulb  is 
completely  filled  with  a  2  per  cent,  solution  of  sodium 
hydroxide  (NaOH)  and  closed  tightly  with  the  thumb. 
The  fluid  is  shaken  thoroughly  with  the  gas  and  allowed 
to  flow  back  and  forth  from  bulb  to  closed  branch  and 
the  reverse  several  times,  to  insure  intimate  contact  of 
the  CO2  with  the  alkali.  Lastly,  before  removing  the 
thumb  all  the  gas  is  allowed  to  collect  in  the  closed  branch, 
so  that  none  may  escape  when  the  thumb  is  removed. 
If  CO2  be  present,  a  partial  vacuum  in  the  closed  branch 
causes  the  fluid  to  rise  suddenly  when  the  thumb  is  re- 
moved. After  allowing  the  layer  of  foam  to  subside 
somewhat  the  space  occupied  by  gas  is  again  measured, 
and  the  difference  between  this  amount  and  that  meas- 

1  Loc.  cit.,  p.  196. 


206  BACTERIOLOGY. 

ured  before  shaking  with  the  sodium  hydroxide  solution 
gives  the  proportion  of  CO2  absorbed.  The  explosive 
character  of  the  residue  is  determined  as  follows  :  the 
cotton  plug  is  'replaced  and  the  gas  from  the  closed 
branch  is  allowed  to  flow  into  the  bulb  and  mix  with 
the  air  there  present.  The  plug  is  then  removed  and 
a  lighted  match  inserted  into  the  mouth  of  the  bulb. 
The  intensity  of  the  explosion  varies  with  the  amount 
of  air  present  in  the  bulb." 

CULTIVATION  WITHOUT  OXYGEN. — As  we  have 
already  learned,  there  is  a  group  of  bacteria  to  which 
the  name  "  anaerobic  organisms  "  has  been  given,  which 
are  characterized  by  inability  to  grow  in  the  presence 
of  free  oxygen.  For  the  cultivation  of  the  members 
of  this  group  a  number  of  devices  are  employed  for 
the  exclusion  of  free  oxygen  from  the  cultures. 

Koch's  method.  Koch  covered  the  surface  of  a  gel- 
atin plate,  which  had  been  previously  inoculated,  with 
a  thin  sheet  of  sterilized  isinglass.  The  organisms 
which  grew  beneath  it  were  supposed  to  develop  with- 
out oxygen. 

Hesse's  method.  Hesse  poured  sterilized  oil  upon  the 
surface  of  a  culture  made  by  stabbing  a  tube  of  gelatin. 
The  growth  that  occurred  along  the  track  of  the  needle 
was  supposed  to  be  anaerobic  in  nature. 

Methods  of  Liborius.  Liborius  has  suggested  two 
useful  methods  for  this  purpose.  One  is  to  nearly  (about 
three-quarters)  fill  a  test-tube  with  gelatin  or  agar-agar, 
which,  after  having  been  sterilized,  is  to  be  kept  in  a 
vessel  of  boiling  water  for  ten  minutes  to  expel  all  air 
from  it.  It  is  then  rapidly  cooled  in  ice-water,  and 
when  between  30°  and  40°  C.,  still  fluid,  is  to  be  inoc- 
ulated and  very  rapidly  solidified.  It  is  then  sealed  in 


CULTIVATION   WITHOUT  OXYGEN. 


207 


the  flame.  Anaerobic  bacteria  develop  only  in  the  lower 
layers  of  the  medium.  In  his  other  method  he  employs 
a  special  tube,  known  as  "  the  Liborius  tube."  Its  con- 
struction is  shown  in  Fig.  40. 


FIG.  40. 


Liborius  tube  for  anaerobic  cultures. 


Through  the  side  tube  hydrogen  is  passed  until  it  re- 
places all  the  air ;  the  contracted  parts,  both  of  the  neck 
of  the  tube  and  the  side  arm,  are  then  sealed  in  the 
flame.1  This  tube  can  be  used  for  either  solid  or  liquid 
media,  but,  owing  to  its  usual  small  capacity,  gives 
better  results  with  fluid  media.  (For  precautions  in 

1  As  the  tubes  come  from  the  maker  the  contracted  parts  marked  x 
in  the  cut  are  usually  so  thick  as  to  render  the  sealing  in  the  flame 
during  the  passage  of  hydrogen  somewhat  troublesome ;  it  is  better 
to  draw  them  out  in  the  flame  quite  thin  before  passing  the  hydrogen 
into  the  tube.  This  makes  the  final  sealing  a  matter  of  no  difficulty. 


208  BACTERIOLOGY. 

using  hydrogen,  see  note  to  FriinkePs  method,  page 
210.) 

Method  of  Buchner.  The  plan  suggested  by  Buchner, 
of  allowing  the  cultures  to  develop  in  an  atmosphere 
robbed  of  its  oxygen  by  pyrogallic  acid,  gives  very  good 
results.  In  this  method  the  culture,  which  is  either  a 
slant-  or  stab-culture  in  a  test-tube,  is  placed — tube, 
cotton  plug,  and  all — into  a  larger  tube,  in  the  bottom 
of  which  have  been  deposited  1  gramme  of  pyrogallic 
acid  and  10  c.c.  of  ^  normal l  caustic-potash  solution. 
The  larger  tube  is  then  tightly  plugged  with  a  rubber 
stopper.  The  oxygen  is  quickly  absorbed  by  the  pyro- 
gallic acid,  and  the  organisms  develop  in  the  remaining 
constituents  of  the  atmosphere,  viz.,  nitrogen,  a  small 
amount  of  CO2,  and  a  trace  of  ammonia. 

Method  of  C.  Frdnkel.  Carl  Frankel  suggests  the 
following  as  a  modification  of  or  substitute  for  the  tube 
of  Liborius  :  the  tube  is  first  inoculated  as  if  it  were 
to  be  poured  as  a  plate  or  rolled  as  an  ordinary  Esmarch 
tube.  The  cotton  plug  is  then  replaced  by  a  rubber 
stopper,  through  which  pass  two  glass  tubes.  These 

1  A  normal  solution  is  one  that  contains  in  a  litre  as  many  grammes 
of  the  dissolved  substance  as  are  indicated  by  its  molecular  equivalent. 
The  equivalent  is  that  amount  of  a  chemical  compound  which  possesses 
the  same  chemical  value  as  does  one  atom  of  hydrogen.  For  example  : 
one  molecule  of  hydrochloric  acid  (HC1)  has  a  molecular  weight  and 
also  an  equivalent  weight  of  36.5 ;  a  molecule  of  this  acid  has  the 
same  chemical  value  as  one  atom  of  hydrogen.  Its  normal  solution  is 
therefore  36.5  grammes  to  the  litre.  On  the  other  hand,  sulphuric  acid 
(H2SO4)  contains  in  each  molecule  two  replaceable  hydrogen  atoms ; 
its  normal  solution  is  not,  therefore,  80  grammes  (itemolecular  weight) 
to  the  litre,  but  that  amount  which  would  be  equivalent  chemically  to 
one  hydrogen  atom,  viz.,  40  grammes  (one-half  its  molecular  weight) 
to  the  litre.  A  normal  solution  of  caustic  potash  contains  as  many 
grammes  to  the  litre  as  the  number  of  its  molecular  weight— 56.1 
grammes  to  the  litre  of  water. 


CULTIVATION  WITHOUT  OXYGEN. 


209 


must  all  have  been  sterilized  in  the  steam  sterilizer 
before  using.  On  the  outer  side  of  the  stopper  these 
two  tubes  are  bent  at  right  angles  to  the  long  axis  of 
the  test-tube  into  which  they  are  to  be  placed,  and  both 
are  slightly  drawn  out  in  a  gas-flame.  Both  of  these 


FIG.  41. 


Frankel's  method  for  the  cultivation  of  anaerobic  bacteria. 

tubes  must  be  provided,  before  sterilization,  with  a 
plug  of  cotton  ;  this  is  to  prevent  the  access  of  foreign 
organisms  to  the  medium  during  manipulations.  At 
the  inner  side  of  the  rubber  stopper — that  is,  the  end 
which  is  to  be  inserted  into  the  test-tube — the  glass 
tubes  are  of  different  lengths :  one  reaches  to  within 
0.5  cm.  of  the  bottom  of  the  test-tube,  the  other  is  cut 
off  flush  with  the  under  surface  of  the  stopper.  The 

14 


210  BACTERIOLOGY. 

outer  end  of  the  longer  glass  tube  is  then  connected 
with  a  hydrogen  generator  and  hydrogen  is  allowed 
to  bubble  through  the  gelatin  (Fig.  41,  A)  in  the  tube 
until  all  contained  air  has  been  expelled  and  its  place 
taken  by  the  hydrogen.1  When  the  hydrogen  has  been 
bubbling  through  the  gelatin  for  about  five  minutes 
(at  least)  one  can  be  reasonably  sure  that  all  oxygen 
has  been  expelled.  The  drawn-out  portions  of  the 
tubes  can  then  be  sealed  in  the  gas-flame  without  fear 
of  an  explosion.  The  protruding  end  of  the  rubber 
stopper  is  then  painted  around  with  melted  paraffin 
and  the  tube  rolled  in  the  way  given  for  ordinary 
Esmarch  tubes.  A  tube  thus  prepared  and  containing 
growing  colonies  is  shown  in  Fig.  41,  B. 

The  development  that  now  occurs  is  in  an  atmos- 
phere of  hydrogen,  all  oxygen  having  been  expelled. 
During  the  operation  the  tube  containing  the  liquefied 

1  Before  beginning  the  experiment  it  is  always  wise  to  test  the  hydro- 
gen— i.  e.,  to  see  that  it  is  free  from  oxygen  and  that  there  is  no  danger 
of  an  explosion,  for  unless  this  be  done  the  entire  apparatus  may  be 
blown  to  pieces  and  a  serious  accident  occur.  The  agents  used  should 
be  pure  zinc  and  pure  sulphuric  acid  of  about  25  to  30  per  cent, 
strength.  With  the  primary  evolution  of  the  gas  the  outlet  of  the 
generator  should  be  closed  and  kept  closed  until  the  gas  reservoir  is 
quite  filled  with  hydrogen.  The  outlet  should  then  be  opened  and  the 
entire  volume  of  gas  allowed  to  escape,  care  being  taken  that  no  flame 
is  in  the  neighborhood.  This  should  be  repeated,  after  which  a  sample 
of  the  hydrogen  generated  should  be  collected  in  an  inverted  test-tube 
in  the  ordinary  way  for  collecting  gases  over  water,  viz.,  by  filling  a 
test-tube  with  water,  closing  its  mouth  with  the  thumb,  inverting  it, 
and  placing  its  mouth  under  water,  when,  after  removing  the  thumb, 
the  water  will  be  kept  in  it  by  atmospheric  pressure.  The  hydrogen 
which  is  flowing  from  the  open  generator  may  be  conducted  to  the  test- 
tube  by  rubber  tubing.  When  the  water  has  been  replaced  test  the  gas 
by  holding  a  flame  near  the  open  mouth  of  the  test-tube.  If  no  explo- 
sion occurs,  the  hydrogen  is  safe  to  use.  Should  there  be  an  explosion, 
the  generation  of  hydrogen  must  be  continued  in  the  apparatus  until 
it  burns  with  a  colorless  flame  when  tested  in  a  test-tube. 


CULTIVATION   WITHOUT  OXYGEN.  211 

gelatin  should  be  kept  in  a  water-bath  at  a  temperature 
sufficiently  high  to  prevent  its  solidifying,  and  at  the 
same  time  not  high  enough  to  kill  the  organisms  with 
which  it  has  been  inoculated. 

One  of  the  obstacles  to  the  successful  performance  of 
this  method  is  the  bubbling  of  the  gelatin,  the  foam 
from  which  will  often  fill  the  exit-tube  and  sometimes 
be  forced  from  it.  This  may  be  obviated  by  reversing 
the  order  of  proceeding,  viz. :  roll  the  Esmarch  tube 
in  the  ordinary  way  with  the  organisms  to  be  studied, 
using  a  relatively  small  amount  of  gelatin,  so  as  to 
have  as  thin  a  layer  as  possible  when  it  is  rolled. 
Then  replace  the  cotton  plug  with  the  sterilized  rubber 
stopper  carrying  the  glass  tubes  through  which  the 
hydrogen  is  to  be  passed,  and  allow  the  hydrogen  to 
flow  through  as  in  the  method  first  given.  The  gas 
now  passes  over  the  gelatin  instead  of  through  it, 
and  consequently  no  bubbling  results.  In  all  other 
respects  the  procedure  is  the  same  as  that  given  by 
Frankel. 

Method  of  Kitasato  and  Weil.  For  favoring  an- 
aerobic conditions  Kitasato  and  Weil  have  suggested 
the  addition  to  the  culture-media  of  some  strong  re- 
ducing-agent.  They  recommend  formic  acid  or  sodium 
formate  in  0.3  to  0.5  per  cent. ;  glucose  in  1.5  to  2  per 
cent. ;  or  blue  litmus  tincture  in  5  per  cent,  by  volume. 
This  is,  of  course,  in  addition  to  an  atmosphere  from 
which  all  oxygen  has  been  expelled.  As  a  reducing- 
agent  for  this  purpose  Theobald  Smith  regards  a  weaker 
solution  of  glucose,  0.3  to  0.5  per  cent.,  as  more  ad- 
vantageous. 

Esmarch's  method.  Esmarch's  plan  is  to  prepare  in 
the  usual  way  a  roll- tube  of  the  organisms ;  subject  it 


212  BACTERIOLOGY. 

to  a  low  temperature,  and  while  quite  cold  fill  it  with 
liquefied  gelatin,  which  is  caused  to  solidify  rapidly. 
In  this  method  the  colonies  develop  along  the  sides  of 
the  tubes,  and  can  be  more  easily  studied  than  when 
they  are  scattered  through  the  gelatin,  as  in  the  method 
of  Liborius. 

By  some  workers  the  oxygen  is  removed  from  the 
culture-medium  by  the  use  of  the  air-pump. 

Many  other  methods  are  employed  for  this  special 
purpose,  but  for  the  beginner  those  given  will  suffice. 

From  what  has  been  said  it  may  be  inferred  that  the 
cultivation  of  anaerobic  bacteria  is  a  simple  matter  and 
attended  with  but  little  difficulty.  Such  an  inference 
will,  however,  be  quickly  abandoned  when  the  beginner 
attempts  this  part  of  his  work  for  the  first  time,  and 
particularly  when  his  efforts  are  directed  toward  the 
separation  of  these  forms  from  other  organisms  with 
which  they  are  associated.  The  presence  of  spore- 
forming,  facultative  anaerobes  in  mixed  cultures  is 
always  to  be  suspected,  and  it  is  this  group  that  renders 
the  task  so  difficult.  At  best  the  work  requires  undi- 
vided attention  and  no  small  degree  of  skill  in  bacterio- 
logical technique. 

INDOL  PRODUCTION. — The  generation  of  products 
other  than  those  that  give  rise  to  alterations  in  the  reac- 
tion of  the  media,  and  whose  presence  may  be  detected 
by  chemical  reactions,  is  now  a  recognized  step  in  the 
identification  of  different  species  of  bacteria.  Among 
these  products  is  one  that  is  produced  by  a  number 
of  organisms,  and  whose  presence  may  easily  be  de- 
tected by  its  characteristic  behavior  when  treated  with 
certain  substances.  I  refer  to  nttroso-indol,  the  reac- 
tions of  which  were  described  by  Beyer  in  1869,  and 


INDOL  PRODUCTION.  213 

the  presence  of  which  as  a  product  of  the  growth  of 
certain  bacteria  has  since  furnished  a  topic  for  consid- 
erable discussion. 

Indol,  the  name  by  which  this  body  is  now  generally 
known,  when  acted  upon  by  reducing-agents  becomes 
of  a  more  or  less  decided  rose  color.  This  body  was 
recognized  as  one  of  the  products  of  growth  of  the 
spirillum  of  Asiatic  cholera  first  by  Poel,  and  a  short 
time  subsequently  by  Bujwid  and  by  Dunham,  and  for 
a  time  was  believed  to  be  peculiarly  characteristic  of 
the  growth  of  this  organism.  It  has  since  been  found 
that  there  are  many  other  bacteria  which  also  possess 
the  property  of  producing  indol  in  the  course  of  their 
development.  It  is  constantly  present  in  putrefying 
matters,  and  is  one  of  the  aromatic  bodies  that  give  to 
faeces  their  characteristic  odor. 

The  methods  employed  for  its  detection  are  as  follows  : 
cultivate  the  organism  for  twenty-four  to  forty-eight 
hours  at  a  temperature  of  37°  C.,  in  the  simple  pep- 
tone solution  known  as  "  Dunham's  solution "  (see 
formula  for  this  medium).  This  solution  is  preferred 
because  its  pale  color  does  not  mask  the  rose  color  of 
the  reaction  when  the  amount  of  indol  present  is  very 
small. 

Four  tubes  should  always  be  inoculated  and  kept 
under  exactly  the  same  conditions  for  the  same  length 
of  time. 

At  the  end  of  twenty-four  or  forty-eight  hours  the 
test  may  be  made.  Proceed  as  follows  :  to  a  tube  con- 
taining 7  c.c.  of  the  peptone  solution,  but  which  has  not 
been  inoculated,  add  10  drops  of  concentrated  sulphuric 
acid.  To  another  similar  tube  add  1  c.c.  of  a  0.01  per 
cent,  solution  of  sodium  nitrite,  and  afterward  10  drops 


214  BACTERIOLOGY. 

of  concentrated  sulphuric  acid.  Observe  the  tubes  for 
five  to  ten  minutes.  No  alteration  in  their  color  ap- 
pears, or  at  least  there  is  no  production  of  a  rose  color. 
They  contain  no  indol. 

Treat  in  the  same  way,  with  the  acid  alone,  two  of 
the  tubes  which  have  been  inoculated.  If  no  rose  color 
appears  after  five  or  ten  minutes,  add  1  c.c.  of  the 
sodium  nitrite  solution.  If  now  no  rose  color  is  pro- 
duced, the  indol  reaction  may  be  considered  as  negative 
— i.  e.,  no  indol  has  been  formed  as  a  product  of  the 
growth  of  the  bacteria. 

If  indol  is  present,  and  the  rose  color  appears  after 
the  addition  of  the  acid  alone,  it  is  plain  that  not  only 
indol  has  been  formed,  but  coincidently  a  reducing- 
body.  This  is  found,  by  proper  means,  to  be  nitrous 
acid.  The  sulphuric  acid  liberates  this  acid  from  its 
salts  and  permits  of  its  reducing  action  being  brought 
into  play. 

If  the  rose  color  appears  only  after  the  addition  of 
both  the  acid  and  the  nitrite  solution,  then  indol  has 
been  formed  during  the  growth  of  the  organisms,  but 
no  nitrites. 

Control  the  results  obtained  by  treating  the  two 
remaining  cultures  in  the  same  way. 

The  test  is  sometimes  made  by  allowing  concentrated 
sulphuric  acid  to  flow  down  the  sides  and  collect  at  the 
bottom  of  the  tube ;  the  reaction  is  then  seen  as  a  rose- 
colored  zone  overlying  the  line  of  contact  of  the  acid  and 
culture-medium.  This  method  is  open  to  the  objection 
that,  if  indol  is  present  in  only  a  very  small  amount, 
the  faint  rose  tint  produced  by  it  is  apt  to  be  masked 
by  a  brown  color  that  results  from  the  charring  action 
of  the  concentrated  acid  on  the  other  organic  matters 


INDOL  PRODUCTION.  215 

in  the  culture-medium,  so  that  its  presence  may  in  this 
way  escape  detection.  In  view  of  this,  Petri  recom* 
mends  the  use  of  dilute  sulphuric  acid.  He  states  that 
when  indol  is  present  the  characteristic  rose  color  ap- 
pears a  little  more  slowly  with  the  dilute  acid,  but  it  is 
more  permanent,  and  there  is  never  any  likelihood  of 
its  presence  being  masked  by  other  color-reactions. 

Muir  and  Ritchie  recommend  the  use  of  ordinary 
fuming  or  yellow  nitric  acid  for  this  test.  In  this 
method  two  or  three  drops  of  the  acid  are  added  to 
the  culture  under  consideration.  If  indol  be  present, 
the  red  color  appears  as  a  result  of  the  reducing  action 
of  the  nitrous  acid  upon  it.  The  defect  in  this  method 
is  that  it  reveals  the  presence  of  only  indol,  and  does 
not  indicate  whether  or  not  reducing-bodies  were  coin- 
cidently  formed  with  the  indol.  As  a  test  for  indol 
alone  it  is  convenient  and  entirely  trustworthy. 

Test  far  nitrites.  For  this  purpose  Lunkewicz  has 
recently  recommended  the  employment  of  Ilosvay's 
modification  of  the  method  of  Griess.  As  reagents 
the  following  solutions  are  employed : 

a.  Naphthylamine 0.1  gramme. 

Distilled  water 20.0  c.c. 

Acetic  acid  (25  per  cent  sol.) 150.0  c.c. 

b.  Sulfanilic  acid 0.5  gramme. 

Acetic  acid  (25  per  cent,  sol.) 150.0  c.c. 

In  preparing  solution  a  the  naphthylamine  is  dis- 
solved in  20  c.c.  of  boiling  water,  filtered,  allowed  to 
cool,  and  mixed  with  the  dilute  acetic  acid.  Solutions 
a  and  6  are  then  mixed.  The  resulting  mixture  should 
be  colorless.  It  is  best  to  prepare  it  as  needed,  though 
if  kept  in  a  closely  stoppered  flask  it  retains  its  virtues 
for  some  time. 


216  BACTERIOLOGY. 

When  added  to  cultures  containing  nitrites,  in  the 
proportion  of  one  volume  of  reagent  to  five  volumes 
of  the  culture,  a  deep-red  color  appears  in  a  few 
seconds.  If  110  nitrites  are  present,  no  color-reaction 
occurs.  In  testing  cultures  always  control  the  results 
by  tests  on  a  portion  of  the  same  medium  not  inocu- 
lated, as  some  of  the  ingredients  of  which  the  medium 
is  composed  may  contain  nitrites.  Lunkewicz  recom- 
mends the  use  of  Merck's  peptone  for  this  test,  claim- 
ing that  nitrites  are  always  to  be  found  in  Witte's 
peptones. 

POINTS  TO  BE  OBSERVED  IN  DESCRIBING  AN  ORGANISM. 

The  following  is  an  outline  of  points  to  be  considered 
in  describing  a  new  organism  or  in  identifying  an 
organism  with  one  already  described : 

1.  Its  source — as  air,  water,  or  soil.     If  found  in  the 
animal  body,  is  it  normally  present  or  only  in  patholog- 
ical conditions? 

2.  Its  form,  size,  mode  of  development,  occurrence 
of  involution-forms  or  other  variations  in  morphology. 
Grouping,  as  in  pairs,  chains,  clumps,  zoogkea;  pres- 
ence   of    capsule ;    development   and    germination   of 
spores ;  arrangement  of  flagella. 

3.  Staining-peculiarities — especially  its  reactions  with 
Gram's  (or  Weigert's  fibrin)  stain,  and  peculiar  or  irreg- 
ular modes  of  staining. 

4.  Motility — to  be  determined  on  very  fresh  cultures 
and  on  cultures  in  different  media. 

5.  Its  relation  to  oxygen — Is  it  aerobic,  anaerobic, 
or   facultative?     Does   it  develop   in   other  gases,   as 
carbonic  acid,  hydrogen,  etc.  ? 


DESCRIBING  AN  ORGANISM.  217 

6.  Both  the  macroscopic  and  microscopic  appearance 
of  its  colonies  on  nutrient  gelatin  and  on  nutrient  agar- 
agar. 

7.  The  appearance  of  its  growth  in  stab-  and  slant- 
cultures  on  gelatin,  agar-agar,  blood-serum,  and  potato. 

8.  The  character  of  its  growth  in  fluid  media,  as  in 
bouillon,  milk,  litmus-milk,  rosolic-acid-peptone   solu- 
tion, and  in  bouillon  containing  glucose. 

9.  Does  it  grow  best   in   acid,  alkaline,  or   neutral 
media  ? 

10.  Is  the  normal  reaction  of  the  medium  altered  by 
its  growth  ?     Is  its  growth  accompanied  by  the  produc- 
tion of  indol  ?     Is  the  indol  associated  with  the  coinci- 
dent production  of  nitrites  ? 

11.  Is  its  growth  accompanied  by  the  production  of 
gas,  as  evidenced  by  the  appearance  of  bubbles  in  the 
media — both  in    media  containing  fermentable   sugars 
and  those  from  which  these  bodies  are  absent?     When 
cultivated  in    sugar-bouillon  in  the  fermentation-tube, 
what  proportion  of  gas  is  evolved  under  known  condi- 
tions ?     How  much  of  this  gas  is  carbonic  acid,  and  how 

O  / 

much  is  explosive? 

12.  At  what  temperature   does  it   thrive   best,  and 
what  are  the  lowest  and  highest  temperatures  at  which 
it  will  develop?     What  is  its  thermal  death-point,  both 
by  steam  and  dry-air  methods  of  determining  this  point? 

13.  What  is  its  behavior  when  exposed  to  chemical 
disinfectants  and  antiseptics?     Does  it  withstand  dry- 
ing and  other  injurious  influences,  both  in  the  vegeta- 
tive and  spore  stages  ?     The  germicidal  activity  of  the 
blood-serum   of  different    animals   may  also   be   tried 
upon  it. 

14.  Its  pathogenic  powers — modes  of  inoculation  by 


218  BACTERIOLOGY. 

which  these  are  demonstrated  ;  quantity  of  material  used 
in  inoculation  ;  duration  of  the  disease  and  its  symp- 
toms; lesions  produced,  and  distribution  of  the  bacteria 
in  the  inoculated  animals  ;  which  animals  are  susceptible 
and  which  immune,  and  the  character  of  its  pathogenic 
activities.  Variations  in  virulence,  and  the  probable 
causes  to  which  they  are  due.  Can  they  be  produced 
artificially  and  at  will  ? 

15.  The  detection  of  specific,  toxic,  and  immunizing 
products  of  growth. 

16.  Its  behavior  when  exposed  to  the  influence  of 
blood-serum  of  animals   immunized  from  it ;   also  its 
behavior  Svhen  mixed  with  serum  from  an  animal  in 
the  acute  stage  of  infection  resulting  from  it.     Are  the 
relations  between  the  organism  and  the  serum  constant 
and  specific? 


CHAPTER  XII. 

Inoculation  of  animals — Subcutaneous  inoculation — Intravenous  in- 
jection— Inoculation  into  the  great  serous  cavities,  and  into  the 
anterior  chamber  of  the  eye — Observation  of  animals  after  inocu- 
lation. 

AFTER  subjecting  an  organism  to  the  methods  of 
study  that  we  have  thus  far  reviewed  there  remains  to 
be  tested  its  action  upon  animals — i.  e.y  to  determine  if 
it  possesses  the  property  of  producing  disease  or  not ; 
and,  if  so,  what  are  the  pathological  results  of  its 
growth  in  the  tissues  of  these  animals,  and  in  what  way 
must  it  gain  entrance  to  the  tissues  in  order  to  produce 
these  results  ?  The  mode  of  deciding  these  points  is  by 
inoculation,  which  is  practised  in  different  ways  accord- 
ing to  circumstances.  Most  commonly  a  bit  of  the 
culture  to  be  tested  is  simply  introduced  beneath  the 
skin  of  the  animal ;  but  in  other  cases  it  may  be  neces- 
sary to  introduce  it  directly  into  the  vascular  or  lym- 
phatic circulation,  or  into  one  or  the  other  of  the  great 
serous  cavities ;  or,  for  still  other  purposes  of  observa- 
tion, into  the  anterior  chamber  of  the  eye,  upon  the  iris. 

SUBCUTANEOUS  INOCULATION  OF  ANIMALS. — The 
animals  usually  employed  in  the  laboratory  for  purposes 
of  inoculation  are  white  mice,  gray  house-mice,  guinea- 
pigs,  rabbits,  and  pigeons. 

For  simple  subcutaneous  inoculation  the  steps  in  the 
process  are  practically  the  same  in  all  cases.  The  hair  or 
feathers  are  to  be  carefully  removed.  If  the  skin  is  very 

219 


220  BACTERIOLOGY. 

dirty,  it  may  be  scrubbed  with  soap  and  water.  Steriliza- 
tion of  the  skin  is  practically  impossible,  so  it  need  not 
be  attempted.  If  the  inoculation  is  to  be  made  by  means 
of  a  hypodermic  syringe,  then  a  fold  of  the  skin  may  be 
lifted  up  and  the  needle  inserted  in  the  usual  way.  If  a 
solid  culture  is  to  be  inoculated,  a  fold  of  skin  may  be 
taken  up  with  forceps  and  a  pocket  cut  into  it  with 
scissors  which  have  previously  been  sterilized.  This 
pocket  must  be  cut  large  enough  to  admit  the  end  of  the 
needle  without  its  touching  the  sides  of  the  opening  as  it 
is  inserted.  Beneath  the  skin  will  be  found  the  super- 
ficial and  deep  connective-tissue  fasciae.  These  must  be 
taken  up  with  sterilized  forceps,  and  with  sterilized  scis- 
sors incised  in  a  way  corresponding  to  the  opening  in  the 
skin.  The  pocket  is  then  to  be  held  open  with  the  for- 
ceps and  the  substance  to  be  inserted  is  introduced  as 
far  under  the  skin  and  fasciae  as  possible,  care  being 
taken  not  to  touch  the  edges  of  the  wound  if  it  can 
be  avoided.  The  edges  of  the  wound  may  then  be 
simply  pulled  together  and  allowed  to  remain.  No 
stitching  or  efforts  at  closing  it  are  necessary,  though  a 
drop  of  collodion  over  the  point  of  operation  may  serve 
to  lessen  contamination. 

During  manipulation  the  animal  must  be  held  still. 
For  this  purpose  special  forms  of  holders  have  been 
devised ;  but  if  an  assistant  is  at  hand,  the  simple  sub- 
cutaneous inoculation  may  be  made  without  the  aid  of 
a  mechanical  holder. 

It  is  at  times,  however,  more  convenient  to  dispense 
with  an  assistant ;  one  of  several  forms  of  apparatus  that 
have  been  devised  for  holding  mice,  guinea-pigs,  rats, 
rabbits,  etc.,  may  then  be  used.  For  small  animals,  such 
as  mice  and  rats,  the  holder  suggested  by  Kitasato  is  very 


SUBCUTANEOUS  INOCULATION  OF  ANIMALS,    221 

useful.    It  is  simply  a  metal  plate  attached  to  a  stand  by  a 
clamped  ball-and-socket  joint,  so  that  it  can  be  fixed  in 


FIG.  42. 


Kitasato's  mouse-holder. 


any  position.     It  is  provided  with  a  spring-clip  at  one 
end  that  holds  the  animal  by  the  skin  of  the  neck,  and 


FIG.  43. 


Holder  for  larger  animals. 

at  the  other  end  with  another  clamp  that  holds  the  tail 
of  the  animal.     This  holder  is  shown  in  Fig.  42.     For 


222 


BACTERIOLOGY. 


larger  animals  the  form  of  holder  shown  in  Fig.  43  is 
commonly  used. 

A  very  simple  and  useful  holder  for  guinea-pigs  con- 
sists of  a  metal  cylinder  of  about  5  cm.  diameter  and 


FIG.  44. 


The  Voges  holder  for  guinea-pigs. 


about  13  cm.  long,  closed  at  one  end  by  a  perforated 
cap  of  either  tin  or  wire  netting.  Along  the  side  of 
this  box  is  a  longitudinal  slit  12  mm.  wide  that  runs  for 
9.5  cm.  from  within  0.5  mm.  of  the  open  extremity  of 
the  cylinder.  The  animal  is  placed  in  such  a  cylinder 


SUBCUTANEOUS  INOCULATION  OF  ANIMALS.    223 

with  its  head  toward  the  perforated  bottom.  It  is  then 
easily  possible  to  make  subcutaneous  inoculation  by 
taking  up  a  bit  of  skin  through  the  slit  in  the  side  of 
the  box,  or  to  make  intraperitoneal  injection  by  drawing 
the  posterior  extremities  slightly  from  the  box  and 
holding  them  steady  between  the  index  and  second 
finger,  as  seen  in  Fig.  44.  It  is  also  very  convenient 
for  use  when  the  rectal  temperature  of  these  small  ani- 
mals is  to  be  taken.  The  manipulations  can  easily  be 
made  without  the  aid  of  an  assistant.  Its  construction 
is  seen  in  Fig.  44.1 

For  ordinary  subcutaneous  inoculations  at  the  root 
of  the  tail  in  mice  a  simple  apparatus  consists  of  a 
piece  of  board  about  7  x  10  cm.  and  2  cm.  thick, 
upon  which  is  tacked  a  hollow  truncated  cone  of  wire 
gauze,  about  6  cm.  long  and  about  1.5  cm.  in  diameter 
at  one  end  and  2  cm.  at  its  other  end.  This  is  tacked 

FIG.  45. 


Mouse-holder,  with  mouse  in  proper  position. 

upon  the  board  in  such  a  position  that  its  long  axis  is 
in  the  long  axis  of  the  board,  being  equidistant  from  its 
sides.  Its  small  end  is  placed  at  the  edge  of  the  board. 
The  mouse  is  taken  up  by  the  tail  by  means  of  a  pair 

1  Centralblatt  fur  Bakteriologie  und  Parasitenkunde,  1895,  Bd.  xviii. 
p.  530. 


224  BACTERIOLOGY. 

of  tongs  and  allowed  to  crawl  into  the  smaller  end  of 
the  wire  cone.  When  so  far  in  that  only  the  root  of 
the  tail  projects  the  animal  is  fixed  in  this  position  by 
a  clamp  and  thumb-screw,  with  which  the  apparatus 
(Fig.  45)  is  provided.  The  animal  usually  remains  per- 
fectly quiet  and  may  be  handled  without  difficulty. 

The  hair  over  the  root  of  the  tail  is  to  be  care- 
fully cut  away  with  scissors  and  a  pocket  cut  through 
the  skin  at  this  point.  The  inoculation  is  then  made 
into  the  loose  tissue  under  the  skin  over  this  part  of  the 
back  in  the  way  that  has  just  been  described.  It  is 
always  best  to  insert  the  needle  some  distance  along  the 
spinal  column,  and  thus  deposit  the  material  as  far  from 
the  surface-wound  as  possible. 

As  the  subcutaneous  inoculation  is  very  simple  and 
takes  only  a  few  moments,  guinea-pigs,  rabbits,  and 
pigeons  may  be  held  by  an  assistant.  The  front  legs  in 
the  one  hand  and  the  hind  legs  in  the  other,  with  the 
animal  stretched  upon  its  back  on  a  table,  is  the  usual 
position  for  the  operation  when  practised  upon  guinea- 
pigs  and  rabbits.  The  point  at  which  the  inoculations 
are  commonly  made  is  in  the  abdominal  wall,  either  to 
the  right  or  left  of  the  median  line  and  about  3  cm. 
distant.  When  pigeons  are  used  they  are  held  with  the 
legs,  tail,  and  ends  of  the  wings  in  the  one  hand,  and 
the  head  and  anterior  portion  of  the  body  in  the  other, 
leaving  the  area  occupied  by  the  pectoral  muscles,  over 
which  the  inoculation  is  to  be  made,  free  for  manipu- 
lation. The  hair  over  the  point  selected  for  the  in- 
oculation should  be  closely  cut  with  scissors  in  the  case 
of  guinea-pigs  and  rabbits,  and  from  a  small  area  the 
feathers  should  be  plucked  in  the  case  of  the  pigeon. 

INJECTION  INTO  THE  CIRCULATION. — It  is  not  in- 


INJECTION  INTO   THE  CIRCULATION.          225 

frequently  desirable  to  inject  the  material  under  consid- 
eration directly  into  the  circulation  of  an  animal.  If 
a  rabbit  is  employed  for  the  purpose,  the  operation  is 
usually  done  upon  one  of  the  veins  in  the  ear.  To  those 
who  have  had  no  practice  with  this  procedure  it  offers  a 
great  many  difficulties ;  but  if  the  directions  which  will 
be  given  are  strictly  observed,  the  greatest  of  these 
obstacles  to  the  successful  performance  of  the  operation 
may  be  overcome. 

When  viewing  the  circulation  in  the  ear  of  the  rabbit 
by  transmitted  light  three  conspicuous  branches  of  the 
main  vessel  (vena  auricularis  posterior)  will  be  seen. 
One  runs  about  centrally  in  the  long  axis  of  the  ear, 
one  runs  along  its  anterior  margin,  and  one  along  its 
posterior  margin.  The  central  branch  (ramus  anterior 
of  the  vena  auricularis  posterior)  is  the  largest  and  most 
conspicuous  vessel  of  the  ear,  and  is,  therefore,  believed 
by  the  inexperienced  to  be  the  branch  into  which  it  would 
appear  easiest  to  insert  a  hypodermic  needle.  This, 
however,  is  fallacious.  This  vessel  lies  very  loosely 
imbedded  in  connective  tissue,  and,  in  efforts  to  intro- 
duce a  needle  into  it,  rolls  about  to  such  an  extent  that 
only  after  a  great  deal  of  difficulty  does  the  experiment 
succeed.  On  the  other  hand,  the  posterior  branch  (ramus 
lateralls  posterior  of  the  vena  auricularis  posterior)  is  a 
very  fine,  delicate  vessel  which  runs  along  the  posterior 
margin  of  the  ear,  and  is  so  firmly  fixed  in  the  dense 
tissues  which  surround  it  that  it  is  prevented  from 
rolling  about  under  the  point  of  the  needle.  The 
further  away  from  the  mouth  of  the  vessel — that  is,  the 
nearer  we  approach  its  capillary  extremity — the  more 
favorable  become  the  conditions  for  the  success  of  the 
operation. 

15 


226  BA  CTERIOL  OGY. 

Select,  then,  the  very  delicate  vessel  lying  quite  close 
to  the  posterior  margin  of  the  ear,  and  make  the  injec- 
tion as  near  to  the  apex  of  the  ear  as  possible.  At 
times  the  vessels  of  the  ear  will  be  found  to  contain 
so  little  blood  that  they  are  hardly  distinguishable, 
making  it  very  difficult  to  introduce  the  needle  into 
them.  This  is  sometimes  overcome  by  pressure  at  the 
root  of  the  ear,  causing  stasis  of  the  blood  and  distention 
of  the  vessels.  A  very  satisfactory  method  of  causing 
the  veins  to  become  prominent  is  to  press  lightly  or 
prick  gently  with  the  point  of  a  needle  the  skin  over 
the  vessel  to  be  used.  In  a  few  seconds,  as  a  result  of 
this  irritation,  the  vessel  will  have  become  distended 
with  blood,  and  readily  distinguishable  from  the  sur- 
rounding tissue ;  it  may  then  be  easily  punctured  by 
the  needle  of  the  syringe.  A  sharp  flick  with  the 
finger  will  often  produce  the  same  result.  The  injection 
is  always  to  be  made  from  the  dorsal  surface  of  the  ear. 

Of  no  less  importance  than  the  selection  of  the  proper 
vessel  is  the  shape  of  the  point  of  the  needle  employed. 
The  hypodermic  needles  as  they  come  from  the 
makers  are  not  suited  at  all  for  this  operation,  because 
of  the  manner  in  which  their  points  are  ground.  If 
one  examine  carefully  the  point  of  a  new  hypodermic 
needle,  it  will  be  seen  that  the  long  point,  instead  of 
presenting  a  flat,  slanting  surface  when  viewed  from  the 
side,  has  a  more  or  less  curved  surface.  Now,  in  efforts 
to  introduce  such  a  needle  into  a  vessel  of  very  small 
calibre  it  is  usually  seen  that  the  point  of  the  needle, 
instead  of  remaining  in  the  vessel,  as  it  would  do  were 
it  straight,  very  commonly  projects  into  the  opposite 
wall ;  and  as  the  needle  is  inserted  further  and  further 
it  is  usually  pushed  through  the  vessel-walls  into  the 


INJECTION  INTO  THE  CIRCULATION.         227 

loose  tissues  beyond,  and  the  material  to  be  injected  is 
deposited  in  these  tissues,  instead  of  into  the  circulation. 
If,  on  the  contrary,  the  slanting  point  of  the  needle  be 
ground  until  its  surface  is  perfectly  flat  when  viewed 
from  the  side,  and  no  curvature  exists,  then  when  once 
inserted  into  a  vessel  it  usually  remains  there,  and  there 
is  no  tendency  to  penetrate  the  opposite  wall.  We  never 
use  a  new  hypodermic  needle  until  its  point  is  carefully 
ground  to  a  perfectly  flat,  slanting  surface  with  no  cur- 
vature whatever. 

These  differences  may  perhaps  be  more  easily  under- 
stood if  represented  diagrammatically.     In  Fig.  46,  a, 


FIG.  46. 
a 


Hypodermic  needles,  magnified,  a.  Improper  point,  b.  Proper  shape  of  point. 

the  needle  has  the  point  usually  seen  when  new.  In  Fig. 
46,  6,  the  point  has  been  ground  to  the  shape  best  suited 
for  this  operation.  The  needles  need  not  be  returned 
to  the  maker.  One  can  grind  them  to  the  shape  desired 
in  a  few  minutes  upon  an  oilstone.  The  size  of  the 
needle  is  that  commonly  employed  by  physicians  for 
subcutaneous  injections  in  human  beings. 

When  the  operation  is  to  be  performed  an  assistant 
holds  the  animal  gently  but  firmly  in  the  crouching 
position  upon  a  table.  If  the  animal  does  not  remain 
quiet,  it  is  be'st  to  wrap  it  in  a  towel,  so  that  only  its 
head  protrudes;  though  in  most  cases  we  have  not 


228  BACTERIOLOGY. 

found  this  necessary,  particularly  if  the  animal  has  not 
been  excited  prior  to  beginning  the  operation. 

The  ear  in  which  the  injection  is  to  be  made  is  to  be 
shaved  clean  of  hair  by  means  of  a  razor  and  soap. 

The  animal  should  be  placed  so  that  the  prepared 
ear  comes  between  the  operator  and  the  source  of  light. 
This  renders  visible  by  transmitted  light  not  only  the 
coarser  vessels  of  the  ear,  but  also  their  finer  branches. 

The  filled  hypodermic  syringe  is  taken  in  one  hand 
and  with  the  other  hand  the  ear  is  held  firmly.  The 
point  of  the  needle  is  then  inserted  through  the  skin 
and  into  the  finest  part  of  the  ramus  posterior,  the  part 
nearest  the  apex  of  the  ear,  where  the  course  of  the 
vessel  is  nearly  straight.  When  the  point  of  the  needle 
is  in  this  vessel  it  gives  to  the  hand  a  sensation  quite 
different  from  that  felt  when  it  is  in  the  midst  of  con- 
nective tissue.  As  soon  as  one  supposes  the  point  of 
the  needle  is  in  the  vessel  a  drop  or  two  of  the  fluid  may 
be  injected  from  the  syringe,  and,  if  his  suspicions  are 
correct,  the  circulation  in  the  small  ramifications  and 
their  anastomoses  will  rapidly  alter  in  appearance — 
i.  e.,  the  circulating  blood  will  be  very  quickly  replaced 
by  the  clear  transparent  fluid  that  is  being  injected.  At 
this  stage  one  must  proceed  very  carefully,  for  some- 
times when  the  needle-point  is  not  actually  in  the  ves- 
sel, but  is  in  the  lymph-spaces  surrounding  it,  an  ap- 
pearance somewhat  similar  is  seen.  This  may  always 
be  differentiated,  however,  by  continuing  the  injection, 
when  the  flow  of  clear  fluid  through  the  vessels  will  not 
only  fail  to  take  the  place  of  the  circulating  blood,  but 
at  the  same  time  a  localized  swelling,  due  to  an  accu- 
mulation of  the  fluid  injected,  will  appear  under  the 
skin  about  the  point  of  the  needle.  The  needle  must 


INJECTION  INTO  THE  CIRCULATION.         229 

then  be  withdrawn  and  inserted  into  the  vessel  at  a 
point  a  little  nearer  its  proximal  end. 

Care  must  be  taken  that  no  air  is  injected. 

The  hypodermic  syringe  and  needle  must,  previous 
to  operation,  have  been  carefully  sterilized  in  the  steam 
sterilizer  or  in  boiling  water.  The  animal  must  be 
kept  under  close  observation  for  about  an  hour  after 
injection. 

The  operation  is  one  that  cannot  be  learned  from 
verbal  description.  It  can  only  be  successfully  per- 
formed after  actual  practice.  If  the  precautions  which 
have  been  mentioned  are  observed,  but  little  difficulty 
in  performing  the  operation  will  be  experienced. 

Its  greater  convenience  and  simplicity,  as  compared 
with  other  methods  for  the  introduction  of  substances 
into  the  circulation,  commend  it  as  a  technical  procedure 
with  which  to  make  one's  self  familiar.  The  animals 
sustain  practically  no  Avound,  they  experience  no  suffer- 
ing— at  least  they  give  no  evidence  of  pain — and  no 
anaesthetic  is  required. 

The  form  of  syringe  best  suited  for  this  operation  is 
of  the  ordinary  design,  but  one  that  permits  of  thorough 
sterilization  by  steam.  It  should  be  made  of  glass  and 
metal,  with  packings  that  may  be  sterilized  by  steam 
without  injury.  The  syringes  commonly  employed  are 
those  shown  in  Fig.  47. 

For  operations  requiring  exact  dosage  experience 
has  led  me  to  prefer  a  syringe  after  the  pattern  of  C, 
in  Fig.  47 — i.  e.y  the  form  commonly  used  by  physi- 
cians. The  reason  for  this  is  as  follows :  in  making 
injections,  either  into  the  circulation  or  under  the  skin, 
there  is  a  certain  amount  of  resistance  to  the  passage  of 
fluid  from  the  needle.  If  one  overcomes  this  resistance 


230 


BACTERIOLOGY. 


by  means  of  a  cushion  of  compressed  air,  as  is  the  case 
in  syringes  A  and  B,  Fig.  47,  the  sudden  expansion 
of  the  air  in  the  body  of  the  syringe  when  resistance  is 
overcome  frequently  causes  a  larger  amount  of  fluid 
to  be  ejected  than  is  desired.  No  such  accident  is  likely 


FIG.  47. 


Forms  of  hypodermic  syringe. 
A.  Koch's  syringe.    B.  Syringe  of  Strohschein.    C.  Overlack's  form. 

to  occur  when  the  fluid  is  forced  from  the  barrel  of  the 
syringe  by  the  head  of  a  close-fitting  piston,  with  no  air 
intervening  between  the  fluid  and  the  head  of  the  piston. 
With  such  an  instrument,  properly  manipulated,  the 
dose  can  always  be  controlled  with  accuracy. 

INOCULATION  INTO  THE  LYMPHATIC  CIRCULATION. 
— Fluid  cultures  or  suspensions  of  bacteria  may  be  in- 
jected into  the  lymphatics  by  way  of  the  testicles.  The 
operation  is  in  no  wise  complicated.  One  simply  plunges 
the  point  of  the  hypodermic  needle  directly  into  the  sub- 
stance of  the  testicle  and  then  injects  the  amount  desired. 
Injections  made  in  this  manner  are  sometimes  followed 
by  interesting  pathological  lesions  of  the  lymphatic 
apparatus  of  the  abdomen. 


l\(t('ULATION  INTO  GREAT  StiROUS  CAVITIES.    231 

INOCULATION  INTO  THE  GREAT  SEROUS  CAVITIES. 
—Inoculation  into  the  peritoneum  presents  no  difficulties 
if  fluids  are  to  be  introduced.  In  this  case  one  makes, 
with  a  pair  of  sterilized  scissors,  a  small  nick  through 
the  skin  down  to  the  underlying  fasciae,  and,  taking  a 
fold  of  the  abdominal  wall  between  the  fingers,  plunges 
the  hypodermic  needle  through  the  opening  just  made 
directly  into  the  peritoneal  cavity.  There  is  little  or  no 
danger  of  penetrating  the  intestines  or  other  internal 
viscera  if  the  puncture  be  made  along  the  median  line 
at  about  midway  between  the  end  of  the  sternum  and 
the  symphysis  pubis.  Though  this  may  seem  a  rude 
method,  it  is  rarely  that  the  intestines  are  penetrated. 
The  object  of  the  primary  incision  is  to  lessen  the 
chances  of  contamination  by  bacteria  located  in  the 
skin,  some  of  which  would  adhere  to  the  needle  if  it 
were  plunged  directly  through  the  skin,  and  might 
complicate  the  results. 

If  solid  substances,  bits  of  tissue,  etc.,  are  to  be  intro- 
duced into  the  peritoneum,  it  becomes  necessary  to  con- 
duct the  operation  upon  the  lines  of  a  laparotomjy. 
The  hair  should  be  shaved  from  a  small  area  over  the 
median  line,  after  which  the  skin  is  to  be  thoroughly 
washed.  A  short  longitudinal  incision  (about  2  cm. 
long)  is  then  to  be  made  in  the  median  line  through  the 
skin  and  down  to  the  fasciae.  Two  subcutaneous 
sutures,  as  employed  by  Halsted,  are  then  to  be  intro- 
duced transversely  to  the  line  of  incision  about  1  cm. 
apart,  and  their  ends  left  loose.  This  particular  sort  of 
suture  does  not  pass  through  the  skin,  but,  instead,  the 
needle  is  introduced  into  the  subcutaneous  tissues  along 
the  edge  of  the  incision.  In  this  case  they  are  to  pass 
into  the  abdominal  cavity  and  out  again,  entering  at  one 


232 


BACTERIOLOGY. 


side  of  the  line  of  incision  and  leaving  at  the  other, 
as  indicated  by  the  solid  and  dotted  lines  in  Fig.  48. 
(The  figure  indicates  the  primary  opening  through  the 
skin.  The  longitudinal  dotted  line  shows  the  opening  to 


FIG.  48. 


Diagram  of  skin  incision  and  sutures  in  laparotomy  on  animals. 

be  made  into  the  abdomen ;  the  transverse  dotted  lines, 
with  their  loose  ends,  represent  the  sutures  as  placed 
in  position  be/ore  the  abdomen  is  opened ;  it  will  be  seen 
that  these  sutures  in  all  cases  pass  through  the  subcuta- 
neous tissues  only  and  do  not  penetrate  the  skin  proper.) 
The  opening  through  the  remaining  layers  may  now 
be  completed ;  the  bit  of  tissue  is  deposited  in  the  peri- 
toneal cavity,  under  precautions  that  will  exclude  all 
else,  the  edges  of  the  wound  drawn  evenly  and  gently 
together  by  tying  the  sutures,  and  the  lines  of  incision 
dressed  with  collodion.  It  should  be  needless  to  say 
that  this  operation  must  be  conducted  under  the  strictest 
precautions,  to  avoid  complications.  All  instruments, 
sutures,  ligatures,  etc.,  must  be  carefully  sterilized  either 


ANIMALS  AFTER  INOCULATION.  233 

in  the  steam  sterilizer  for  twenty  minutes,  or  by  boiling 
in  2  per  cent,  sodium  carbonate  solution  for  ten  min- 
utes ;  the  hands  of  the  operator,  though  they  should  not 
touch  the  wound,  must  be  carefully  cleansed,  and  the 
material  to  be  introduced  into  the  abdomen  should  be 
handled  with  only  sterilized  instruments. 

Inoculation  into  the  pleural  cavity  is  much  less  fre- 
quently required — in  fact,  it  is  not  a  routine  method. 
It  is  not  easy  to  enter  the  pleural  cavity  with  a  hypo- 
dermic needle  without  injuring  the  lung,  and  it  is  rare 
that  conditions  call  for  the  introduction  of  solid  parti- 
cles into  this  locality. 

Inoculation  into  the  anterior  chamber  of  the  eye  is  per- 
formed by  making  a  puncture  through  the  cornea  just 
in  front  of  its  junction  with  the  sclerotic,  the  knife  being 
passed  into  the  anterior  chamber  in  a  plane  parallel  to 
the  plane  of  the  iris.  By  the  aid  of  a  fine  pair  of  for- 
ceps the  bit  of  tissue  is  passed  through  the  opening  thus 
made  and  is  deposited  upon  the  iris,  where  it  is  allowed 
to  remain,  and  where  its  pathogenic  activities  upon  the 
iris  can  be  conveniently  studied.  It  is  a  mode  of  inoc- 
ulation of  very  limited  application,  and  is  therefore  but 
rarely  practised.  It  was  employed  in  the  classical 
experiments  of  Cohnheim  in  demonstrating  the  infec- 
tious nature  of  tuberculous  tissues,  tuberculosis  of  the 
iris  being  the  constant  result  of  the  introduction  of 
tuberculous  tissue  into  the  anterior  chamber  of  the  eye 
of  rabbits. 

OBSERVATION  OF  ANIMALS  AFTER  INOCULATION. 
— After  either  of  these  methods  of  inoculation,  particu- 
larly when  unknown  species  of  bacteria  are  being  tested, 
the  animal  is  to  be  kept  under  constant  observation  and 
all  deviations  from  the  normal  are  to  be  carefully  noted 


234 


BACTERIOLOGY. 


— as,   for  instance,  elevation  of  temperature  ;   loss  of 
weight ;  peculiar  position  in  the  cage  ;  loss  of  appetite ; 


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ANIMALS  AFTER  INOCULATION. 


235 


.-it(  of  inoculation,  etc.     If  death  ensue  in  from  two  to 
four  days,  it  may  reasonably  be  expected  that  at  autopsy 


evidence  of  either  acute  septic  or  toxic  processes  will  be 
found.  It  sometimes  occurs,  however,  that  inoculation 
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236 


BACTERIOLOGY. 


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ANIMALS  AFTER  INOCULATION. 


237 


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weighed  daily,  always   at  about  the   same  hour  and 
always  about  midway  between  the  hours  of  feeding ; 


238  BA  CTERIOLOG  Y. 

at  the  same  time  its  temperature,  as  indicated  by  a 
thermometer  placed  in  the  rectum,  is  to  be  recorded.1 
By  comparison  of  these  daily  observations  the  ob- 
server is  aided  in  determining  the  course  the  infection 
is  taking. 

Too  much  stress  must  not,  however,  be  laid  upon 
moderate  and  sudden  daily  fluctuations  in  either  tem- 
perature or  weight,  as  it  is  a  common  observation  that 
presumably  normal  animals  when  confined  in  cages  and 
fed  regularly  often  present  very  striking  temporary 
gains  and  losses  in  weight,  often  amounting  to  50  or 
100  grammes  in  twenty-four  hours,  even  in  animals 
whose  total  weight  may  not  exceed  500  or  600  gammes; 
similarly  unexplai  liable  rises  and  falls  of  temperature, 
often  as  much  as  a  degree  from  one  day  to  another,  are 
seen.  Such  fluctuations  have  apparently  no  bearing 
upon  the  general  condition  of  the  animal,  but  are  prob- 
ably due  to  transient  causes,  such  as  overfeeding  or 
scarcity  of  food,  improper  feeding,  lack  of  exercise, 
excitement,  fright,  etc. 

The  accompanying  charts  (Figs.  49,  50,  51,  52)  will 
serve  to  illustrate  some  of  these  points.  The  animals, 
two  rabbits  and  two  guinea-pigs,  were  taken  at  random 
from  among  stock  animals  and  placed  each  in  a  clean 
cage,  the  kind  used  for  animals  under  experiment,  and 
kept  under  as  good  general  conditions  as  possible.  For 
the  first  week  the  rabbits  received  each  100  grammes 
of  green  food  (cabbage  and  turnips)  daily,  and  the 
guinea-pigs  30  grammes  each  of  the  same  food.  During 


1  The  thermometer  must  be  inserted  into  the  rectum  beyond  the 
grasp  of  the  sphincter,  otherwise  pressure  upon  its  bulb  by  contraction 
of  this  muscle  may  force  up  the  mercurial  column  to  a  point  higher 
than  that  resulting  from  the  actual  body-temperature. 


ANIMALS  AFTER  INOCULATION.  239 

the  second  week  this  daily  amount  of  food  was  doubled  ; 
during  the  third  week  it  was  quadrupled ;  and  for  the 
fourth  and  fifth  weeks  they  each  received  an  excess  of 
food  daily,  consisting  of  green  vegetables  and  grains 
(oats  and  corn).  By  reference  to  the  charts  sudden 
diurnal  fluctuations  in  weight  will  be  observed  that 
do  not  correspond  in  all  instances  with  scarcity  or  suf- 
ficiency of  food.  With  the  rabbits  there  is  a  gradual 
loss  of  weight  with  the  smaller  amounts  of  food,  which 
losses  are  not  totally  recovered  as  the  food  is  increased. 
With  the  guinea-pigs  there  is  likewise  at  first  a  loss ; 
but  after  a  short  time  the  weight  remains  tolerably  con- 
stant, and  is  not  so  conspicuously  affected  by  the  increase 
in  food  as  one  might  expect.  From  the  recorded  tem- 
peratures one  sees  the  peculiar  fluctuations  mentioned. 
To  just  what  they  are  due  it  is  impossible  to  say.  It 
is  manifest  that  the  normal  temperature  of  these  animals, 
if  we  can  speak  of  a  normal  temperature  for  animals 
presenting  such  fluctuations,  is  about  a  degree  or  more, 
Centigrade,  higher  than  that  of  human  beings.  The 
animals  from  which  these  charts  were  made  were  not 
inoculated,  nor  were  they  subjected  to  any  operative 
procedures  whatever,  the  only  deviations  from  normal 
conditions  being  the  variations  in  the  daily  amount  of 
food  given. 

In  certain  instances,  however,  there  will  be  noticed 
a  constant  tendency  to  diminution  in  weight,  notwith- 
standing the  daily  fluctuations,  and  after  a  time  a  con- 
dition of  extreme  emaciation  may  be  reached,  the 
animal  often  being  reduced  to  from  50  to  60  per  cent, 
of  its  original  weight.  In  other  cases,  after  inoculations 
to  which  the  animal  is  not  susceptible,  rabbits  in  par- 
ticular, if  properly  fed,  will  frequently  gain  steadily  in 


240  BACTERIOLOGY. 

weight.  The  condition  of  progressive  emaciation  just 
mentioned  is  conspicuously  seen  after  intravenous  inoc- 
ulation of  rabbits  with  cultures  of  bacillus  typhi  ab- 
dominalis  and  of  bacterium  coli  commune,  referred  to 
in  the  chapter  on  the  latter  organism,  and  if  looked 
for  will  doubtless  be  seen  to  follow  inoculation  with 
other  organisms  capable  of  producing  chronic  forms 
of  infection,  but  which  are  frequently  considered  non- 
pathogenic  because  of  their  inability  to  induce  acute 
conditions.  Not  infrequently  in  chronic  infections  there 
may  be  hardly  any  marked  and  constant  temperature- 
variations  until  just  before  death,  when  sometimes  there 
will  be  a  rise  and  at  other  times  a  fall  of  temperature. 
In  the  majority  of  cases,  however,  one  must  be  very 
cautious  as  to  the  amount  of  stress  laid  upon  changes 
in  weight  and  temperature,  for  unless  they  are  progres- 
sive or  continuous  in  one  or  another  direction  they  may 
have  little  or  no  significance  as  indicating  the  existence 
or  absence  of  disease. 


CHAPTER    XIII. 

Post-mortem  examination  of  animals — Bacteriological  examination  of 
the  tissues — Disposal  of  tissues  and  disinfection  of  instruments 
after  the  examination — Study  of  tissues  and  exudates  during  life. 

DURING  bacteriological  examination  of  the  tissues 
of  dead  animals  certain  precautions  must  be  rigidly 
observed  in  order  to  arrive  at  correct  conclusions. 

The  autopsy  should  be  made  as  soon  as  possible 
after  death.  If  delay  cannot  be  avoided,  the  animal 
should  be  kept  on  ice  until  the  examination  can  be 
made,  otherwise  decomposition  sets  in,  and  the  sapro- 
phytic  bacteria  now  present  may  interfere  with  the 
accuracy  of  results.  When  the  autopsy  is  to  be  made 
the  animal  is  first  inspected  externally,  and  all  visible 
lesions  noted.  It  is  then  to  be  fixed  upon  its  back 
upon  a  board  with  nails  or  tacks.  The  four  legs  and 
the  end  of  the  nose,  through  which  the  tacks  are  driven, 
are  to  be  moderately  extended.  Plates  are  now  to  be 
made  from  the  site  of  inoculation,  if  this  is  subcuta- 
neous. The  surfaces  of  the  thorax  and  abdomen  are 
then  to  be  moistened  to  prevent  the  fine  hairs,  dust, 
etc.,  from  floating  about  in  the  air  and  interfering  with 
the  work.  An  incision  is  then  made  through  the  skin 
from  the  chin  to  the  symphysis  pubis.  This  is  only 
a  skin  incision,  and  does  not  reach  deeper  than  the 
muscles.  It  is  best  done  by  first  making  with  a  scalpel 
an  incision  just  large  enough  to  permit  of  the  intro- 
duction of  one  blade  of  a  blunt-pointed  scissors.  It  is 

16  241 


242  BACTERIOLOGY. 

then  completed  with  the  scissors.  The  whole  of  the 
skin  is  now  to  be  carefully  dissected  away,  not  only 
from  the  abdomen  and  thorax,  but  from  the  axillary, 
inguinal,  and  cervical  regions,  and  the  fore  and  hind 
legs  as  well.  It  is  then  pinned  flat  upon  the  board 
so  as  to  keep  it  as  far  from  the  abdomen  and  thorax  as 
possible,  for  it  is  from  the  skin  that  the  chances  of 
contamination  are  greatest. 

It  now  becomes  necessary  to  proceed  very  carefully. 
All  incisions  from  this  time  on  are  to  be  made  only 
through  surfaces  that  have  been  sterilized.  The  sterili- 
zation is  best  accomplished  by  the  use  of  a  broad-bladed 
table-knife  that  has  been  heated  in  a  gas-flame.  The 
blade,  made  quite  hot,  is  to  be  held  upon  the  region  of 
the  linea  alba  until  the  tissues  of  that  region  begin  to 
burn ;  it  is  then  held  transversely  to  this  line  over  about 
the  centre  of  the  abdomen,  thus  making  two  sterilized 
tracks,  through  which  the  abdomen  may  be  opened  by 
a  crucial  incision.  The  sterilization  thus  accomplished 
is,  of  course,  directed  only  against  organisms  that  may 
have  fallen  upon  the  surface  from  without,  and  there- 
fore it  need  not  extend  deep  down  through  the  tissues. 
In  the  same  way  two  burned  lines  may  be  made  from 
either  extremity  of  the  transverse  line  up  to  the  top  of 
the  thorax. 

With  hot  scissors  the  central  longitudinal  incision 
extending  from  the  point  of  the  sternum  to  the  geni- 
talia  is  to  be  made  without  touching  the  internal  vis- 
cera. The  abdominal  wall  must  therefore  be  held  up 
during  the  operation  with  sterilized  forceps  or  hooks. 
The  cross-incision  is  made  in  the  same  way.  When 
this  is  completed  an  incision  through  the  ribs  with  a 
pair  of  heavy,  sterilized  scissors  is  made  along  the 


POST-MORTEM  EXAMINATION  OF  ANIMALS.  243 

scorched  tracks  on  either  side  of  the  thorax.  After  this 
the  whole  anterior  wall  of  the  thorax  may  easily  be 
lifted  up,  and  by  severing  the  connections  with  the 
diaphragm  it  may  be  completely  removed.  When  this 
is  done  and  the  abdominal  flaps  laid  back,  the  contents 
of  both  cavities  are  to  be  inspected  and  their  condition 
noted  without  disturbing  them. 

After  this  the  first  steps  to  be  taken  are  to  prepare 
plates  or  Esmarch  tubes  from  the  blood,  liver,  spleen, 
kidneys,  and  any  exudates  that  may  exist.  This  is  best 
done  as  follows :  Heat  a  scalpel  quite  hot  and  apply  it 
to  a  small  surface  of  the  organ  from  which  cultures  are 
to  be  made.  Hold  it  upon  the  organ  until  the  surface 
directly  beneath  is  visibly  scorched.  Then  remove  it, 
heat  it  again,  and  while  quite  hot  insert  its  point  through 
the  capsule  of  the  organ.  Into  the  opening  thus  made 
insert  a  sterilized  platinum  loop,  made  of  wire  a  little 
heavier  than  that  commonly  employed.  Project  this 
deeply  into  the  tissues  of  the  organ ;  by  twisting  it 
about  enough  material  from  the  centre  of  the  organ 
can  be  obtained  for  making  the  cultures. 

As  the  resistance  offered  by  the  tissue  is  sometimes 
too  great  to  permit  of  puncture  with  the  ordinary  wire 
loop,  Nuttall l  has  devised  for  the  purpose  a  platinum- 
wire  spear  which  possesses  great  advantage  over  the 
loop.  It  has  the  form  seen  in  Fig.  53.  It  is  easily 
made  by  beating  a  piece  of  heavy  platinum  wire  into  a 
spear-head  at  one  end,  and  perforating  this  with  a  small 
drill,  as  seen  in  the  cut.  It  is  attached  by  the  other  end 
to  either  a  metal  or  glass  handle,  preferably  the  former. 
It  can  readily  be  thrust  into  the  densest  of  the  soft  tissues, 

1  Centralbatt  fur  Bakteriologie  und  Parasitenkunde,  1892,  Bd.  xi. 
p.  538. 


244  BACTERIOLOGY. 

and  by  twisting  it  about  after  its  introduction  particles 
of  the  tissue  sufficient  for  examination  are  withdrawn 
in  the  eye  of  the  spear-head. 

FIG.  53. 


Nuttall's  platinum  spear  for  use  at  autopsies. 

Cultures  from  the  blood  are  usually  made  from 
one  of  the  cavities  of  the  heart,  which  is  always  punct- 
ured at  a  point  which  has  been  burned  in  the  way 
given. 

In  addition  to  cultures,  cover-slips  from  the  site  of 
inoculation,  from  each  organ,  and  from  any  exudates  that 
may  be  present  must  be  made.  These,  however,  are 
prepared  after  the  materials  for  the  cultures  have  been 
obtained.  They  need  not  be  examined  immediately, 
but  may  be  placed  aside,  under  cover,  on  bits  of  paper 
upon  which  the  name  of  the  organ  from  which  they  were 
prepared  is  written. 

When  the  autopsy  is  complete  and  the  gross  appear- 
ances have  been  carefully  noted,  small  portions  of  each 
organ  are  to  be  preserved  in  95  per  cent,  alcohol  for 
subsequent  examination.  Throughout  the  entire  au- 
topsy it  must  be  borne  in  mind  that  all  cultures, 
cover-slips,  and  tissues  must  be  carefully  labelled, 
not  only  with  the  name  of  the  organ  from  which 
they  originate,  but  with  the  date,  designation  of  the 
animal,  etc.,  so  that  an  account  of  their  condition 
after  closer  study  may  be  subsequently  inserted  in  the 
protocol. 

The  cover-slips  are  now  to  be  stained,  mounted,  and 


POST-MORTEM  EXAMINATION  OF  ANIMALS.   245 

examined  microscopically,  and  the  results  carefully 
noted. 

The  same  care  with  regard  to  noting,  labelling, 
etc.,  should  be  exercised  in  the  subsequent  study  of 
the  cultures  and  the  hardened  tissues,  which  are  to  be 
stained  and  subjected  to  microscopic  examination.  The 
results  of  microscopic  study  of  the  cover-slip  prepara- 
tions and  of  those  obtained  by  cultures  should  in  most 
cases  correspond,,  though  it  not  rarely  occurs  that  bac- 
teria are  present  in  such  small  numbers  in  the  tissues 
that  their  presence  may  be  overlooked  microscopically, 
and  still  they  may  appear  in  the  cultures. 

If  the  autopsy  has  been  performed  in  the  proper 
way,  with  the  precautions  given,  and  sufficiently  soon 
after  death,  the  results  of  the  bacteriological  exami- 
nation should  be  either  negative  or  the  organisms 
which  are  isolated  should  be  in  pure  cultures.  This  is 
particularly  the  case  with  cultures  made  from  the  inter- 
nal viscera. 

Both  the  cover-slips  and  cultures  made  from  the 
point  of  inoculation  are  apt  to  contain  a  variety  of 
organisms. 

If  the  organism  obtained  in  pure  culture  from  the 
internal  viscera,  or  those  predominating  at  the  point  of 
inoculation  of  the  animal,  have  caused  its  death,  then 
subsequent  inoculation  of  pure  cultures  of  this  organism 
into  the  tissues  of  a  second  animal  should  produce  sim- 
ilar results. 

When  the  autopsy  is  quite  finished  the  remains  of 
the  animal  should  be  burned;  all  instruments  subjected 
to  either  sterilization  by  steam  or  boiling  for  fifteen 
minutes  in  a  1  to  2  per  cent,  soda  solution ;  and  the 
board  upon  which  the  animal  was  tacked,  as  well  as  the 


246  BACTERIOLOGY. 

tacks,  towels,  dishes,  and  all  other  implements  used  at 
the  autopsy,  be  sterilized  by  steam.  All  cultures, 
cover-slips,  and,  indeed,  all  articles  likely  to  have 
infectious  material  upon  them,  must  be  thoroughly 
sterilized  as  soon  as  they  are  of  no  further  service. 

What  has  been  said  with  regard  to  the  study  of  dead 
tissues  obtained  at  autopsy  applies  equally  well  to  the 
bacteriological  study  of  tissues  and  exudates  obtained 
during  life.  In  the  latter  case,  however,  certain  pre- 
cautions are  always  to  be  observed.  In  the  first  place, 
it  is  desirable  to  obtain  the  materials  under  aseptic  pre- 
cautions, care  being  taken  that  no  disinfectant  fluids 
are  mixed  with  them.  They  should  be  subjected  to 
study  as  soon  as  possible  after  removal  from  the  body. 
In  the  case  of  tissues  that  cannot  be  examined  on  the 
spot,  they  should  be  placed  in  a  sterile  Petri  dish  or 
in  a  stoppered,  sterile,  wide-mouthed  bottle  and  taken 
at  once  to  the  laboratory.  The  surface  should  then  be 
seared  with  a  hot  knife  and  an  incision  through  the 
seared  area  into  the  centre  made  with  a  knife  that  has 
been  sterilized  and  allowed  to  cool.  From  the  depths 
of  this  incision  enough  material  may  be  obtained  for 
microscopic  examination  and  for  the  preparation  of 
cultures.  Fluid  exudates  that  must  be  taken  to  the 
laboratory  should  be  collected  in  either  a  sterile  test- 
tube,  or,  better,  in  a  sterile  capillary  tube  that  is 
subsequently  sealed  at  both  ends  in  a  gas-flame. 
When  bacteriological  examination  of  the  blood  dur- 
ing life  is  required,  it  is  customary  to  obtain  the  neces- 
sary sample  of  blood  by  pricking  the  skin.  It  must 
be  remembered,  in  this  connection,  that  the  skin  usu- 
ally contains  a  number  of  species  of  bacteria  that 


STUDY  OF  TISSUES  DURING  LIFE.  247 

are  of  no  pathological  significance  and  have  nothing 
to  do  with  the  disease  from  which  the  individual 
may  be  suffering.  It  is  manifestly  essential  to  ex- 
clude these.  It  is  not  possible  to  exclude  them  cer- 
tainly and  completely  under  all  circumstances,  without 
a  more  or  less  elaborate  procedure ;  but  an  effort  to  do 
so  should  always  be  made.  -As  a  rule,  the  greater  num- 
ber of  them  may  be  removed  from  the  skin  by  careful 
washing  with  warm  water  and  soap  and  a  sterile  brush, 
after  which  the  skin  should  be  rinsed  with  alcohol  and 
allowed  to  dry  spontaneously.  The  drop  of  blood  may 
then  be  obtained  from  the  skin  thus  cleaned  by  a  prick 
with  a  sharp,  sterilized  lancet.  The  presence  in  the 
cultures  of  a  staphylococcus,  growing  slowly,  with 
white  colonies,  is  a  frequent  experience,  and  does  not 
necessarily  imply  that  this  organism  bears  an  etiological 
relation  to  the  disease  from  which  the  individual  may  be 
suffering  (see  staphylococcus  epideiinidis  albus,  page  268). 
In  the  study  of  many  of  the  common  diseases, 
notably  the  exanthemata,  both  at  autopsy  and  during 
life,  by  the  methods  above  outlined,  the  investigation 
often  yields  negative  results,  and  yet  there  is  every 
reason  for  believing  these  diseases  to  be  dependent  for 
their  existence  upon  invasion  of  the  body  by  some  form 
or  another  of  living  micro-organisms,  capable  of  growth 
in  the  tissues  and  susceptible  of  being  transmitted  from 
individual  to  individual,  either  directly  or  indirectly. 
In  this  connection  it  is  appropriate  to  call  attention 
to  the  novel  and  important  technical  procedures  that 
have  been  employed  by  Nocard  and  Roux l  in  their 
investigations  conducted  in  the  Pasteur  Institute  at 
Paris.  In  the  course  of  their  studies  upon  the  pleuro- 

1  Nocard  and  Eoux :  Annales  de  1'Iiistitut  Pasteur,  April  25,  1898. 


248  BACTERIOLOGY. 

pneumonia  of  cattle,  a  disease  in  which  all  investigators 
had  hitherto  failed  to  detect  by  either  microscopic  or 
culture-methods  any  species  of  bacteria  that  might 
reasonably  be  regarded  as  etiologically  related  to  the 
disease,  they  detected  a  group  of  bodies,  apparently 
bacteria,  of  such  infinitesimally  small  dimensions  as 
entirely  to  escape  detection  by  the  usual  methods  of 
examination. 

The  results  of  Nocard  and  Roux  were  obtained  both 
through  the  adoption  of  special  methods  of  cultivation 
and  the  use  of  very  high  amplifying  powers  for  micro- 
scopic examination.  The  method  of  cultivation  was  that 
suggested  in  1896  by  Metchnikoff,  Roux,  and  Salimbeni,1 
and  is  essentially  as  follows :  very  thin-walled,  small 
sacs  of  collodion  are  sterilized  by  steam,  filled  with 
bouillon,  inoculated  with  the  exudate  or  tissue  to  be 
tested,  sealed  with  sterile  collodion,  and  placed  in  the 
abdominal  cavity  of  an  animal  —  rabbit,  guinea-pig, 
chicken,  dog,  sheep,  or  calf,  as  the  case  may  require. 
The  wall  of  the  collodion  sac  is  impermeable  to  bac- 
teria or  to  leucocytes,  but  is  an  osmotic  membrane 
through  which  fluids  and  some  of  their  dissolved 
contents  readily  diifuse.  This  diffusion  supplies  the 
bacteria  within  the  sacs  with  such  matters  from  the 
living  fluids  of  the  animal  as  are  apparently  essential  to 
their  development,  while  at  the  same  time  the  bacteria 
develop  uninterruptedly,  being  protected  by  the  collodion 
membrane  from  the  antagonistic  action  of  the  fixed  and 
wandering  cells  of  the  tissues.  After  a  period  of  from  a 
few  days  to  several  months  the  animal  is  sacrificed,  and 
the  sac  removed  from  the  peritoneal  cavity  and  its  con- 

1  Metchnikoff,  Eoux,  and  Salimbeni:  Annales  de  1'Institut  Pasteur, 
1896,  p.  257. 


STUDY  OF  TISSUES  DURING  LIFE.  249 

tents  subjected  to  microscopic  examination.  This  latter 
part  of  the  work  was  unsatisfactory  when  conducted  with 
the  usual  combinations  of  lenses  employed  in  bacterio- 
logical work.  Satisfactory  examinations  could  only  be 
made  by  the  use  of  very  high  magnifying  powers,  about 
2000  diameters,  and  unusually  brilliant  illumination. 
When  conducted  under  these  conditions  the  sacs  inoc- 
ulated with  matters  from  the  pulmonary  exudates  of 
pleuro-pneumonia  were  found  to  contain  numerous  mo- 
tile points  or  dots  of  such  extremely  small  size  that  it 
was  often  impossible  to  decide  as  to  their  exact  form. 
Control-sacs  not  inoculated,  but  kept  in  the  peritoneal 
cavity  of  the  same  canal,  manifestly  under  similar  con- 
ditions, did  not  reveal  the  presence  of  the  minute  bodies. 

In  referring  at  length  to  this  investigation  it  is  not 
my  purpose  to  discuss  the  object  of  it,  but  only  to 
direct  attention  to  this  novel  technique,  which  seems 
capable  of  much  wider  application.  There  is  a  group 
of  common  maladies,  such  as  measles,  scarlet  fever, 
smallpox,  etc.,  of  the  etiology  of  which  we  know  noth- 
ing, and  on  which  it  has  hitherto  been  impossible 
to  shed  important  light  by  the  usual  bacteriological 
procedures.  Nocard  and  Roux  have  apparently  re- 
vealed to  us  a  world  of  micro-organisms  whose  existence 
has  hitherto  been  unsuspected,  and  it  is  not  unreason- 
able to  suppose  that  it  is  among  this  group  that  we  are 
to  seek  for  the  causative  agents  of  many  specific  dis- 
eases whose  etiology  is  as  yet  obscure.1 

It  is  proper  also  to  call  attention  to  the  work  of 
Adami  upon  progressive  portal  cirrhosis,  in  which  he 
too,  through  the  use  of  very  high  magnifying  lenses, 

1  An  excellent   review  of  the  paper  of  Nocard  and  Roux  is  to  be 
found  in  the  Philadelphia  Medical  Journal  of  June  11,  1898. 


250  BACTERIOLOGY. 

has  found  an  innnitesimally  small  micro-organism  in 
Pictou  cattle-disease,  "the  main  feature  of  which  is 
extensive  cirrhosis  of  the  liver  accompanied  by  swelling 
of  the  periportal  and  retroperitoneal  lymph-glands.  In 
a  certain  number  of  cases  of  hepatic  cirrhosis  of  human 
beings  Adami  further  believes  that  he  has  found  a  very 
minute  micro-organism  that  may  possibly  stand  in  causal 
relation  to  the  process.1 

1  Adami :  Montreal  Medical  Journal,  July,  1898. 


APPLICATION    OF  THE   METHODS    OF 
BACTERIOLOGY. 


CHAPTER    XIV. 

To  obtain  material  with  which  to  begin  work. 

EXPOSE  to  the  air  of  an  inhabited  room  a  slice  of 
freshly  steamed  potato  or  a  bit  of  slightly  moistened 
bread  upon  a  plate  for  about  one  hour.  Then  cover  it 
with  an  ordinary  water-glass,  place  it  in  a  warm  spot 
(temperature  not  to  exceed  that  of  the  human  body 
— 37.5°  C.),  and  allow  it  to  remain  undisturbed.  In 
from  twenty-four  to  thirty-six  hours  there  will  be  seen 
upon  the  cut  surface  of  the  bread  or  potato  small, 
round,  oval,  or  irregularly  round  patches  which  present 
various  appearances.  These  differences  in  macroscopic 
appearance  are  due  in  some  cases  to  the  presence  or 
absence  of  color;  in  others  to  a  higher  or  lower 
degree  of  moisture ;  in  some  instances  a  patch  will  be 
glistening  and  smooth,  while  its  neighbor  may  be  dull 
and  rough  or  wrinkled;  here  will  appear  an  island 
regularly  round  in  outline,  and  there  an  area  of 
irregular,  ragged  deposit.  All  these  gross  appear- 
ances are  of  .value  in  aiding  us  to  distinguish  between 
these  colonies — for  colonies  they  are,  and  under  the 
same  conditions  the  organisms  composing  each  of  them 
will  always  produce  growth  of  exactly  the  same  ap- 
pearance. It  was  just  such  an  experiment  as  this, 

251 


252  BACTERIOLOGY. 

accidentally  performed,  that  suggested  to  Koch  a  means 
of  separating  and  isolating  in  pure  cultures  the  com- 
ponent individuals  from  mixtures  of  bacteria,  and  it 
was  from  this  observation  that  the  methods  of  cultiva- 
tion on  solid  media  were  evolved. 

If,  without  molesting  these  objects,  we  continue  the 
observations  from  day  to  day,  we  shall  notice  changes 
in  the  colonies,  due  to  the  growth  and  multiplication  of 
the  individuals  composing  them.  In  some  cases  the 
colonies  will  always  retain  their  sharply  cut,  round,  or 
oval  outline,  and  will  increase  but  little  in  size  beyond 
that  reached  after  forty-eight  to  seventy-two  hours; 
whereas  others  will  spread  rapidly  and  quickly  overrun 
the  surface  upon  which  they  are  growing,  and,  indeed, 
grow  over  the  smaller,  less  rapidly  developing  colonies. 
In  a  number  of  instances,  if  the  observation  be  con- 
tinued long  enough,  many  of  these  rapidly  growing 
colonies  will,  after  a  time,  lose  their  lustrous  and  smooth 
or  regular  surface  and  will  show  here  and  there  eleva- 
tions, which  will  continue  to  appear  until  the  whole 
surface  takes  on  a  wrinkled  appearance.  Again,  bub- 
bles may  be  seen  scattered  through  the  colonies.  These 
are  due  to  the  escape  of  gas  resulting  from  fermentation, 
which  the  organisms  bring  about  in  the  medium  upon 
which  they  are  growing.  Sometimes  peculiar  odors  due 
to  the  same  cause  will  be  noticed. 

Note  carefully  all  these  changes  and  appearances,  as 
they  must  be  employed  subsequently  in  identifying  the 
individual  organisms  from  which  each  colony  on  the 
medium  has  developed. 

If  now  we  examine  these  colonies  upon  the  bread  or 
potato  with  a  hand-lens  of  low  magnifying  power,  we 
will  be  enabled  to  detect  differences  not  noticeable  to 


MATERIAL    WITH  WHICH  TO  BEGIN  WORK.   253 

the  naked  eye.  In  a  few  cases  we  may  still  see  nothing 
more  than  a  smooth,  non-characteristic  surface ;  while 
in  others  minute,  sometimes  regularly  arranged  tiny 
corrugations  may  be  observed.  In  one  colony  they  may 
appear  as  tolerably  regular  lines,  radiating  from  a  cen- 
tral spot ;  and  again  they  may  appear  as  concentric 
rings ;  and  if  by  the  methods  which  have  been  de- 
scribed we  obtain  from  these  colonies  their  individual 
components  in  pure  culture,  we  shall  see  that  this 
characteristic  arrangement  in  folds,  radii,  or  concentric 
rings,  or  the  production  of  color,  is  constant  under 
normal  conditions. 

So  much  for  the  simplest  naked-eye  experiment  that 
can  be  made  in  bacteriology,  and  which  serves  to  furnish 
the  beginner  with  material  upon  which  to  commence 
his  studies.  It  is  not  necessary  at  this  time  for  him  to 
burden  his  mind  with  names  for  these  organisms ;  it  is 
sufficient  for  him  to  recognize  that  they  are  mostly  of 
different  species  and  that  they  possess  characteristics 
which  will  enable  him  to  differentiate  the  one  from  the 
other. 

In  order  now  for  him  to  proceed  it  is  necessary  that 
he  should  have  familiarized  himself  with  the  methods 
by  which  his  media  are  prepared  and  the  means  em- 
ployed in  sterilizing  them  and  retaining  them  sterile — 
i.  €.,  of  preventing  the  access  of  foreign  germs  from 
without — otherwise  his  efforts  to  obtain  and  retain  his 
organisms  as  pure  cultures  will  be  in  vain. 

EXPOSURE  AND  CONTACT. — Make  a  number  of  plates 
from  bits  of  silk  used  for  sutures,  after  treating  them 
as  follows : 

Place  some  of  the  pieces  (about  5  centimetres  long) 
in  a  sterilized  test-tube,  and  sterilize  them  by  steam 


254  BACTERIOLOGY. 

for  one  hour,  At  the  end  of  the  sterilization  remove 
one  piece  with  sterilized  forceps  and  allow  it  to  brush 
against  your  clothing,  then  make  a  plate  from  it ;  draw 
another  piece  across  a  table  and  then  plate  it.  Sus- 
pend three  or  four  pieces  upon  a  sterilized  wire  hook 
and  let  them  hang  for  twenty  minutes  free  in  the  air, 
being  sure  that  they  touch  nothing  but  the  hook ;  then 
plate  them  separately. 

Note  the  results. 

In  what  way  do  these  experiments  differ  and  how 
can  the  differences  be  explained  ? 

Expose  to  the  air  six  Petri  dishes  into  which  either 
sterilized  gelatin  or  agar-agar  has  been  poured  and 
allowed  to  solidify ;  allow  them  to  remain  exposed  for 
five,  ten,  fifteen,  twenty,  twenty-five,  and  thirty  min- 
utes in  a  room  where  no  one  is  at  work.  Treat  a  sec- 
ond set  in  the  same  way  in  a  room  where  several  persons 
are  moving  about.  Be  careful  that  nothing  touches 
them  and  that  they  are  exposed  only  to  the  air.  Each 
dish  must  be  carefully  labelled  with  the  time  of  its 
exposure. 

Do  they  present  different  results  ?  What  is  the  rea- 
son for  this  difference  ? 

Which  predominate — colonies  resulting  from  the 
growth  of  bacteria,  or  those  from  common  moulds  ? 

How  do  you  account  for  this  condition  ? 

Sprinkle  a  little  fine  dust  over  the  surface  of  a  plate 
of  sterile  gelatin  or  agar-agar ;  examine  the  dust-par- 
ticles with  the  microscope  immediately  after  depositing 
them  on  the  medium,  and  again  after  eighteen  to  twenty- 
four  hours.  What  differences  do  you  detect  ? 


CHAPTER  XV. 

Various  experiments  in  sterilization  by  steam  and  by  hot  air. 

PLACE  in  one  of  the  openings  in  the  cover  of  the 
steam  sterilizer  an  accurate  thermometer;  when  the 
steam  has  been  streaming  for  a  minute  or  two  the  ther- 
mometer will  register  100°  C.  Wrap  in  a  bundle  of 
towels  or  rags  or  pack  tightly  in  cotton  a  maximum 
(self-registering)  thermometer ;  let  this  thermometer  be 
in  the  centre  of  a  bundle  large  enough  to  quite  fill  the 
chamber  of  the  sterilizer.  At  the  end  of  a  few  minutes' 
exposure  to  the  streaming  steam  remove  it ;  it  will  be 
found  to  indicate  a  temperature  of  100°  C. 

Closer  study  of  the  penetration  of  steam  has  taught 
us,  however,  that  the  temperature  found  at  the  centre 
of  such  a  mass  may  sometimes  be  that  of  the  air  in  the 
meshes  of  the  material,  and  not  that  of  steam,  and  for 
this  reason  the  sterilization  at  that  point  may  not  be 
complete,  because  hot  air  at  100°  C.  has  not  the  ster- 
ilizing properties  that  steam  has  at  the  same  temperature. 
It  is  necessary,  therefore,  that  this  air  should  be  ex- 
pelled from  the  meshes  of  the  material  and  its  place 
taken  by  the  steam  before  sterilization  is  complete.  This 
is  insured  by  allowing  the  steam  to  stream  through  the 
substances  a  few  minutes  before  beginning  to  calculate 
the  time  of  exposure.  There  is  as  yet  no  absolutely 
sure  means  of  saying  that  the  temperature  at  the  centre 
of  the  mass  is  that  of  hot  air  or  of  steam,  so  that  the 
exact  length  of  time  that  is  required  for  the  expulsion 

255 


256  BACTERIOLOGY. 

of  the  air  from  the  meshes  of  the  material  cannot  be 
given. 

Determine  if  the  maximum  thermometer  indicates  a 
temperature  of  100°  C.  at  the  centre  of  a  moist  bundle 
in  the  same  way  as  when  a  dry  bundle  was  employed. 

To  about  50  c.c.  of  bouillon  add  about  1  gramme 
of  chopped  hay,  and  allow  it  to  stand  in  a  warm  place 
for  twenty-four  hours.  At  the  end  of  this  time  it  will 
be  found  to  contain  a  great  variety  of  organisms.  Con- 
tinue the  observation,  and  ultimately  a  pellicle  will  be 
seen  to  form  on  the  surface  of  the  fluid.  This  pellicle 
is  made  up  of  rods  which  grow  as  long  threads  in 
parallel  strands.  In  many  of  these  rods  glistening 
spores  will  be  seen.  After  thoroughly  shaking,  filter 
the  mass  through  a  fine  cloth  to  remove  coarser  parti- 
cles. 

Pour  into  each  of  several  test-tubes  about  10  c.c.  of 
the  filtrate.  Allow  one  tube  to  remain  undisturbed  in  a 
warm  place.  Place  another  in  the  steam  sterilizer  for 
five  minutes ;  a  third  for  ten  minutes ;  a  fourth  for  one- 
half  hour ;  a  fifth  for  one  hour. 

At  the  end  of  each  of  these  exposures  inoculate  a 
tube  of  sterilized  bouillon  from  each  tube.  Likewise 
make  a  set  of  plates  or  Esmarch  tubes  upon  both  gel- 
atin and  agar-agar  from  each  tube,  and  note  the  results. 
At  the  same  time  prepare  a  set  of  plates  or  Esmarch 
tubes  on  agar-agar  and  on  gelatin  from  the  tube  which 
has  not  been  exposed  to  the  action  of  the  steam. 

The  plates  or  tubes  from  the  unmolested  tube  will 
present  colonies  of  a  variety  of  organisms ;  separate  and 
study  these. 

Those  from  the  tube  which  has  been  sterilized  for 


STERILIZATION  BY  STEAM  AND  HOT  AIR.  257 

five  minutes  will  present  colonies  in  moderate  numbers ; 
Imt,  as  a  rule,  they  will  represent  but  a  single  organ- 
ism. Study  this  organism  in  pure  cultures. 

The  same  may  be  predicted  for  the  tube  which  has 
been  heated  for  ten  minutes,  though  the  colonies  will  be 
fewer  in  number. 

The  thirty-minute  tube  may  or  may  not  give  one  or 
two  colonies  of  the  same  organism. 

The  tube  which  has  been  heated  for  one  hour  is 
usually  sterile. 

The  bouillon  tubes  from  the  first  and  second  tubes 
which  were  heated  will  usually  show  the  presence  of 
only  one  organism — the  bacillus  which  gave  rise  to  the 
pellicle- formation  in  the  original  mixture.  This  organ- 
ism is  bacillus  subtilis.  It  is  especially  adapted  to  the 
study  of  those  various  degrees  of  resistance  to  heat  that 
spore-forming  bacteria  exhibit  at  different  stages  of  their 
development. 

Inoculate  about  100  c.c.  of  sterilized  bouillon  with 
a  very  small  quantity  of  a  pure  culture  of  this  organism, 
and  allow  it  to  stand  in  a  warm  place  for  about  six 
hours.  Now  subject  this  culture  to  the  action  of  steam 
for  five  minutes ;  it  will  be  seen  that  sterilization,  as  a 
rule,  is  complete. 

Treat  in  the  same  way  a  second  flask  of  bouillon, 
inoculated  in  the  same  way  with  the  same  organism, 
but  after  having  stood  in  a  warm  place  for  from  forty- 
eight  to  seventy-two  hours — that  is,  until  spores  have 
formed — and  it  will  be  found  that  sterilization  is  not 
complete  :  the  spores  of  this  organism  have  resisted  the 
action  of  steam  for  five  minutes. 

To  determine  if  sterilization  is  complete  always  resort 
to  the  culture  methods,  as  the  macroscopic  and  micro- 

17 


258  BACTERIOLOGY. 

scopic  methods  are  deceptive ;  cloudiness  of  the  media 
or  the  presence  of  bacteria  microscopically  does  not 
always  signify  that  the  organisms  possess  the  property 
of  life. 

Inoculate  in  the  same  way  a  third  flask  of  bouillon 
with  a  very  small  drop  from  one  of  the  old  cultures  upon 
which  the  pellicle  has  formed  ;  mix  it  well  and  subject 
it  to  the  action  of  steam  for  two  minutes ;  then  place  it 
to  one  side  for  from  twenty  to  twenty-four  hours,  and 
again  heat  for  two  minutes ;  allow  it  to  stand  for  another 
twenty-four  hours,  and  repeat  the  process  on  the  third 
day.  No  pellicle  will  be  formed,  and  yet  spores  were 
present  in  the  original  mixture,  and,  as  we  have  seen, 
the  spores  of  this  organism  are  not  killed  by  an  exposure 
of  five  minutes  to  steam.  How  can  this  result  be  ac- 
counted for? 

Saturate  several  pieces  of  cotton  thread,  each  about  2 
cm.  long,  in  the  original  decomposed  bouillon,  and  dry 
them  carefully  at  the  ordinary  temperature  of  the  room  ; 
then  at  a  little  higher  temperature — about  40°  C. — to 
complete  the  process.  Regulate  the  temperature  of  the 
hot-air  sterilizer  for  about  100°  C.,  and  subject  several 
pieces  of  this  infected  and  dried  thread  to  this  temper- 
ature for  the  same  lengths  of  time  that  we  exposed  the 
same  organisms  in  bouillon  to  the  steam,  viz.,  five,  ten, 
thirty,  and  sixty  minutes.  At  the  end  of  each  of  these 
periods  remove  a  bit  of  thread,  and  prepare  a  set  of 
plates  or  Esmarch  tubes  from  it.  Are  the  results  anal- 
ogous to  those  obtained  when  steam  was  employed  ? 

Increase  the  temperature  of  the  dry  sterilizer  and 
repeat  the  process.  Determine  the  temperature  and 
time  necessary  for  the  destruction  of  these  organisms 
by  dry  heat.  These  threads  should  not  be  simply 


STERILIZATION  BY  STEAM  AND  HOT  AIR.  259 

laid  upon  the  bottom  of  the  sterilizer,  but  should  be 
suspended  from  SL  glass  rod,  which  may  be  placed  inside 
the  oven,  extending  across  its  top  from  side  to  side. 

Place  several  of  the  infected  threads  in  the  centre  of 
a  bundle  of  rags.  Subject  this  to  a  temperature  neces- 
sary to  sterilize  the  threads  by  the  dry  method.  Treat 
another  similar  bundle  to  sterilization  by  steam.  In 
what  way  do  the  results  of  the  two  processes  differ? 


CHAPTER    XVI. 

Suppuration— The  staphylococcus  pyogenes  aureus— Staphylococcus 
pyogenes  albus  and  citreus — Streptococcus  pyogenes — Staphylo- 
coccus epidermidis  albus — Bacillus  pyocyaneus — General  remarks. 

STAPHYLOCOCCUS   PYOGENES   AUREUS. 

PREPARE  a  set  of  plates  of  agar-agar  from  the  pus 
of  an  acute  abscess  or  boil  that  has  been  opened  under 
antiseptic  precautions.  Care  must  be  taken  that  none 
of  the  antiseptic  used  gains  access  to  the  culture-tubes, 
otherwise  its  antiseptic  effect  may  be  operative  and  the 
development  of  the  organisms  interfered  with.  It  is 
best,  therefore,  to  take  a  drop  of  the  pus  upon  a  plati- 
num-wire loop  after  it  has  been  flowing  for  a  few  sec- 
onds ;  even  then  it  must  be  taken  from  the  mouth  of 
the  incision  and  before  it  has  run  over  the  surface  of  the 
skin.  At  the  same  time  prepare  two  or  three  cover- 
slips  from  the  pus. 

Microscopic  examination  of  these  slips  will  reveal  the 
presence  of  a  large  number  of  pus-cells,  both  multi- 
nucleated  and  with  horseshoe-shaped  nuclei,  some 
threads  of  disintegrated  and  necrotic  connective  tissue, 
and,  lying  here  and  there  throughout  the  preparation, 
small  round  bodies  which  will  sometimes  appear  singly, 
sometimes  in  pairs,  and  frequently  will  be  seen  grouped 
together  somewhat  like  clusters  of  grapes.  (See  Fig. 
54.)  They  stain  readily  and  are  commonly  located  in 
the  material  between  the  pus-cells ;  very  rarely  they 
may  be  seen  in  the  protoplasmic  body  of  the  cell. 

260 


SUPPURATION.  261 

(Compare  the  preparation  with  a  similar  one  made 
from  the  pus  of  gonorrhea.  (See  Fig.  57.)  In  what 
way  do  the  two  preparations  differ,  the  one  from  the 
other?) 

FIG.  54. 


V 


. 

' 

Preparation  from  pus,  showing  pus-cells,  A,  and  staphylococci,  C. 

After  twenty-four  hours  in  the  incubator  the  plates 
will  be  seen  to  be  studded  here  and  there  with  yellow 
or  orange-colored  colonies,  which  are  usually  round, 
moist,  and  glistening  in  their  naked-eye  appearances. 
When  located  in  the  depths  of  the  medium  they  are 
commonly  seen  to  be  lozenge  or  whetstone  in  shape, 
while  often  they  appear  as  irregular  stars  with  blunt 
points,  and  again  as  irregularly  lobulated  dense  masses. 
In  structure  they  are  conspicuous  for  their  density. 
Under  the  low  objective  they  appear,  when  on  the  sur- 
face, as  coarsely  granular,  irregularly  round  patches, 
with  more  or  less  ragged  borders  and  a  dark  irregular 
central  mass,  which  has  somewhat  the  appearance  of 
masses  of  coarser  clumps  of  the  same  material  as  that- 
composing  the  rest  of  the  colony.  Microscopically, 
these  colonies  are  composed  of  small  round  cells,  irreg- 


262  BACTERIOLOGY. 

ularly  grouped  together.  They  are  in  every  way  of 
the  same  appearance  as  those  seen  upon  the  original 
cover-slip  preparation. 

Prepare  from  one  of  these  colonies  a  pure  stab-culture 
in  gelatin.  After  thirty-six  to  forty-eight  hours  lique- 
faction of  the  gelatin  along  the  track  of  the  needle, 
most  conspicuous  at  its  upper  end,  will  be  observed. 
As  growth  continues  the  liquefied  portion  becomes  more 
or  less  of  a  stocking-shape,  and  gradually  widens  at  its 
upper  end  into  an  irregular  funnel.  This  will  continue 
until  the  whole  of  the  gelatin  in  the  tube  eventually 
becomes  fluid.  There  can  always  be  noticed  at  the 
bottom  of  the  liquefying  portion  an  orange-colored  or 
yellow  mass  composed  of  a  number  of  the  organisms 
which  have  sunk  to  the  bottom  of  the  fluid. 

On  potato  the  growth  is  quite  luxuriant,  appearing  as 
a  brilliant,  orange-colored  layer,  somewhat  tabulated 
and  a  little  less  moist  than  when  growing  upon  agar- 
agar. 

It  does  not  produce  fermentation  with  gas-production. 

It  belongs  to  the  group  of  facultative  aerobes. 

In  milk  it  rapidly  brings  about  coagulation  with  acid 
reaction. 

It  is  not  motile,  and  being  of  the  family  of  micro- 
cocci  does  not  form  endogenous  spores.  It  possesses, 
however,  a  marked  resistance  to  detrimental  agencies. 

In  bouillon  it  causes  a  diffuse  clouding,  and  after  a 
time  presents  a  yellow  sedimentation. 

This  organism  is  the  commonest  of  the  pathogenic 
bacteria  with  which  we  shall  meet.  It  is  staphylo- 
coeeus  pyogenes  aureus,  and  is  the  organism  most  fre- 
quently concerned  in  the  production  of  acute,  circum- 
scribed, suppurative  inflammations.  It  is  almost  every- 


STAPHYLOCOCCUS  PYOGENES  AUREUS.       263 

where  present,  and  is  the  organism  that  is  of  con- 
tinuous annoyance  to  the  surgeon. 

In  studying  its  effects  upon  lower  animals  a  number 
of  points  are  to  be  remembered.  While  it  is  the  etio- 
logical  factor  in  the  production  of  most  of  the  suppu- 
rntive  processes  in  man,  still  it  is  with  no  little  difficulty 
that  these  conditions  can  be  reproduced  in  lower  ani- 
mals. Its  subcutaneous  introduction  into  their  tissues 
does  not  always  result  in  abscess-formation,  and  when  it 
does  there  seems  to  have  been  some  coincident  interfer- 
ence with  the  circulation  and  nutrition  of  these  tissues 
which  renders  them  less  able  to  resist  its  inroads.  When 
introduced  into  the  great  serous  cavities  of  the  lower 
animals  its  presence  is  likewise  not  always  followed  by 
the  production  of  inflammation.  If  the  abdominal 
cavity  of  a  dog,  for  example,  be  carefully  opened  so  as 
to  make  as  slight  a  wound  as  possible,  and  no  injury  be 
done  to  the  intestines,  large  quantities  of  bouillon  cult- 
ures or  watery  suspensions  of  this  organism  may  be, 
and  repeatedly  have  been  introduced  into  the  peritoneum 
without  the  slightest  injury  to  the  animal.  On  the  con- 
trary, if  some  substance  which  acts  as  a  direct  irritant 
to  the  intestines — such,  for  example,  as  a  small  bit  of 
potato  upon  which  the  organisms  are  growing — be  at 
the  same  time  introduced,  or  the  intestines  be  mechani- 
cally injured,  so  that  there  is  a  disturbance  in  their  cir- 
culation, then  the  introduction  of  these  organisms  is 
promptly  followed  by  acute  and  fatal  peritonitis.  (Hal- 
sted.1) 

On  the  other  hand,  the  results  which  follow  their  in- 
troduction into  the  circulation  are  practically  constant. 

1  Halsted :  The  Johns  Hopkins  Hospital  Reports.     Report  in  Sur- 
gery, No.  1,  1891,  vol.  ii.  No.  5,  pp.  301-303. 


264  EA  CTEEIOL  OGY. 

If  one  inject  into  the  circulation  of  the  rabbit  through 
a  vein  of  the  ear,  or  in  any  other  way,  from  0.1  to 
0.3  c.c.  of  a  bouillon  culture  or  watery  suspension  of 
a  virulent  variety  of  this  organism,  a  fatal  pyaemia 
always  follows  in  from  two  and  one-half  to  three  days. 
A  few  hours  before  death  the  animal  is  frequently  seen 
to  have  severe  convulsions.  Now  and  then  excessive 
secretion  of  urine  is  noticed.  The  animal  may  appear 
in  moderately  good  condition  until  from  eight  to  ten 
hours  before  death.  At  the  autopsy  a  typical  picture 
presents :  the  voluntary  muscles  are  seen  to  be  marked 
here  and  there  by  yellow  spots,  which  average  the  size 
of  a  flaxseed,  and  are  of  about  the  same  shape.  They 
lie  usually  with  their  long  axis  running  longitudinally 
between  the  muscle-fibres.  As  the  abdominal  and  tho- 
racic cavities  are  opened  the  diaphragm  is  often  seen  to 
be  studded  with  them.  Frequently  the  pericardial  sac 
is  distended  with  a  clear  gelatinous  fluid,  and  almost 
constantly  the  yellow  points  are  seen  in  the  myocar- 
dium. The  kidneys  are  rarely  without  them ;  here 
they  appear  on  the  surface  as  isolated  yellow  points, 
or,  again,  are  seen  as  conglomerate  masses  of  small 
yellow  points  which  occupy,  as  a  rule,  the  area  fed 
by  a  single  vessel.  If  one  makes  a  section  into  one 
of  these  yellow  points,  it  will  be  seen  to  extend  deep 
down  through  the  substance  of  the  kidney  as  a  yellow, 
wedge-shaped  mass,  the  base  of  the  wedge  being  at  the 
surface  of  the  organ. 

It  is  very  rare  that  these  abscesses — for  abscesses  the 
yellow  points  are,  as  we  shall  see  when  we  come  to  study 
them  more  closely — are  found  either  in  the  liver,  spleen, 
or  brain ;  their  usual  location  being,  as  said,  in  the  kid- 
ney, myocardium,  and  voluntary  muscles. 


COVER-SLIPS  AND  SECTIONS.  265 

These  minute  abscesses  contain  a  dry,  cheesy,  necrotic 
centre,  in  which  the  staphylococci  are  present  in  large 
numbers,  as  may  be  seen  upon  cover-slips  prepared  from 
them.  This  organism  may  also  be  obtained  in  pure 
culture  from  these  suppurating  foci. 

Preserve  in  Miiller's  fluid  and  in  alcohol  duplicate 
bits  of  all  the  tissues  in  which  the  abscesses  are  located. 
When  these  tissues  are  hard  enough  to  cut  sections 
should  be  made  through  the  abscess-points  and  the  his- 
tological  changes  carefully  studied. 

MICROSCOPIC  STUDY  OF  COVER-SLIPS  AND  SECTIONS. 
—In  cover-slip  preparations  this  organism  stains  readily 
with  the  ordinary  dyes.  In  tissues,  however,  it  is  best 
to  employ  some  method  by  means  of  which  contrast- 
stains  may  be  utilized,  and  the  location  and  grouping  of 
the  organisms  in  the  tissues  rendered  more  conspicuous. 
When  stained,  sections  of  tissues  containing  these  small 
abscesses  present  the  following  appearances  : 

To  the  naked  eye  will  be  seen  here  and  there  in  the 
section,  if  the  abscesses  are  very  numerous,  small,  darkly 
stained  areas  which  range  in  size  from  that  of  a  pin- 
point up  to  those  having  a  diameter  of  from  1  to  2  mm. 
These  points,  when  in  the  kidney,  may  be  round  or  oval 
in  outline  ;  or  may  appear  wedge-shaped,  with  the  base 
of  the  wedge  toward  the  surface  of  the  organ.  The 
differences  in  shape  depend  frequently  upon  the  direction 
in  which  the  section  has  been  made  through  the  kidney. 
In  the  muscles  they  are  irregularly  round  or  oval. 

When  quite  small  they  appear,  in  stained  sections,  to 
the  naked  eye,  as  simple,  round  or  oval,  darkly  stained 
points ;  but  when  they  are  in  a  more  advanced  stage  a 
pale  centre  can  usually  be  made  out. 

When  magnified  they  appear  in  the  earliest  stages  as 


266  BACTERIOLOGY. 

minute  aggregations  of  small  cells,  the  nuclei  of  which 
stain  intensely.  Almost  always  evidences  of  progress- 
ing necrosis  can  be  seen  about  the  centre  of  these  cell- 
accumulations.  The  normal  structure  of  the  cells  of 
the  tissues  is  more  or  less  destroyed ;  there  is  seen 
a  granular  condition  due  to  cell-fragmentation  ;  at  dif- 
ferent points  about  the  centre  of  this  area  the  tissue 
appears  cloudy  and  the  tissue-cells  do  not  stain  read- 
ily. Round  about  and  through  this  spot  are  seen 
the  nuclei  of  pus-cells,  many  of  which  are  undergoing 
disintegration.  In  the  smallest  of  these  beginning  ab- 
scesses the  staphylococci  are  to  be  seen  scattered  about 
the  centre  of  the  necrotic  tissue ;  but  in  a  more  advanced 
stage  they  are  commonly  seen  massed  together  in  very 
large  numbers  in  the  form  commonly  referred  to  as 
emboli  of  micrococei. 

The  localized  necrosis  of  the  tissues  which  is  seen  at 
the  centre  of  the  abscess  is  the  direct  result  of  the 
action  of  a  poison  produced  by  the  bacteria,  and  repre- 
sents the  starting-point  for  all  abscess-formations. 

When  the  process  is  further  developed  the  different 
parts  of  the  abscess  are  more  easily  detected.  They 
then  present  in  sections  somewhat  the  following  condi- 
tions :  at  the  centre  can  be  seen  a  dense,  granular  mass 
which  stains  readily  with  the  basic  aniline  dyes,  and 
when  highly  magnified  is  found  to  be  made  up  of 
staphylococci.  Sometimes  the  shape  of  this  mass  of 
staphylococci  corresponds  to  that  of  the  capillary  in 
which  the  organisms  became  lodged  and  developed. 
Immediately  about  the  embolus  of  cocci  the  tissues  are 
seen  to  be  in  an  advanced  stage  of  necrosis.  Their 
structure  is  almost  completely  destroyed,  though  the 
destruction  is  seen  to  be  more  advanced  in  some  of  the 


LESS  COMMON  PYOGENIC  ORGANISMS.        267 

elements  of  the  tissues  than  in  others.  As  we  approach 
the  periphery  of  this  faintly  stained  necrotic  area  it 
becomes  marked  here  and  there  with  granular  bodies, 
irregular  in  size  and  shape,  which  stain  in  the  same 
way  as  do  the  nuclei  of  the  pus-cells  and  represent  the 
result  of  disintegration  going  on  in  these  cells. 

Beyond  this  area  we  come  upon  a  dense,  deeply  stained 
zone,  consisting  of  closely  packed  pus-cells ;  of  granular 
detritus  resulting  from  destructive  processes  acting  upon 
these  cells ;  and  of  the  normal  cellular  and  connective- 
tissue  elements  of  the  part.  Here  and  there  through 
this  zone  will  be  seen  localized  areas  of  beginning  death 
of  the  tissues.  This  zone  gradually  fades  away  into 
the  healthy  surrounding  tissues.  It  constitutes  the  so- 
called  "  abscess-wall." 

Such  is  the  picture  presented  by  the  miliary  abscess 
when  produced  experimentally  in  the  rabbit,  and  it  cor- 
responds from  beginning  to  end  with  the  pathological 
changes  which  accompany  the  formation  of  larger  ab- 
scesses in  the  tissues  of  human  beings. 

From  these  small  abscesses  in  the  tissues  of  the 
rabbit  staphylococcus  pyogenes  aureus  may  again  be 
obtained  in  pure  culture,  and  will  present  identically 
the  same  characteristics  that  were  possessed  by  the  cult- 
ure with  which  the  animal  was  inoculated. 

THE   LESS    COMMON    PYOGENIC    ORGANISMS. 

The  pus  of  an  acute  abscess  in  the  human  being 
may  sometimes  contain  other  organisms  beside  staphylo- 
coccus pyogenes  aureus.  Staphylococcus  pyogenes  albus 
and  riti-eus  may  be  found.  The  colonies  of  the  former 
are  white,  those  of  the  latter  are  lemon-color.  With 
these  exceptions  they  are  in  all  essential  cultural  peculi- 


268  BACTERIOLOGY. 

arities  similar  to  staphylococcus  aureus.  As  a  rule,  they 
are  not  virulent  for  animals,  and  when  they  do  pos- 
sess pathogenic  properties  it  is  in  a  much  lower  degree 
than  is  commonly  the  case  with  the  golden  staphy- 
lococcus. Streptococcus  pyogenes  is  also  sometimes 
present.  The  commonest  of  the  pyogenic  organisms, 
however,  is  that  just  described,  viz.,  staphylococcus 
pyogenes  aureus.  An  organism  that  is  almost  univer- 
sally present  in  the  skin,  and  is  often  concerned  in  pro- 
ducing mild  forms  of  inflammation,  is  staphylococcus 
epidermidis  albus  (Welch),  an  organism  that  may  read- 
ily be  confused  with  staphylococcus  albus.  It  is  distin- 
guished from  the  latter  by  the  slowness  with  which  it 
liquefies  gelatin  and  by  the  comparative  absence  of 
pathogenic  properties  when  injected  into  the  circulation 
of  rabbits.  Welch  regards  this  organism  as  a  variety 
of  staphylococcus  pyogenes  albus. 

STREPTOCOCCUS  PYOGENES.  —  From  a  spreading 
phlegmonous  inflammation  prepare  cover-slips  and  cult- 
ures. What  is  the  predominating  organism  ?  Does  it 
appear  in  the  form  of  regular  clusters  like  those  of 
grapes,  or  have  its  individuals  a  definite,  regular  ar- 
rangement ?  (See  Fig.  55.)  Are  its  colonies  like  those 
of  staphylococcus  pyogenes  aureus  ? 

Isolate  this  organism  in  pure  cultures.  In  these  cult- 
ures it  will  be  found  on  microscopic  examination  to 
present  an  arrangement  somewhat  like  a  chain  of  beads. 
(Fig.  56.) 

Determine  its  peculiarities  and  describe  them  accu- 
rately. They  should  be  as  follows : 

Upon  microscopic  examination  a  micrococcus  should 
be  found,  but  differing  in  its  arrangement  from  the 
staphylococci  just  described.  The  single  cells  are  not 


LESS  COMMON  PYOGENIC  ORGANISMS.        269 

scattered  irregularly  or  arranged  in  clumps  similar  to 
bunches  of  grapes,  but  are  joined  together  in  chains  like 
strands  of  beads.  These  strands  are  sometimes  regular 

FIG.  55. 


Streptococcus  pyogenes  in  pus. 


in  the  arrangement  and  size  of  the  individual  cells  com- 
posing them,  but  more  commonly  certain  irregular  groups 
may  be  seen  in  them.  Here  they  appear  as  if  two  or 


..-'"  /    J         -:  -.  : 

'  ..  —  -O   ,•  .......  <, 


Streptococcus  pyogenes. 


three  cells  have  fused  together  to  form  a  link,  so  to  speak, 
in  the  chain,  that  is  somewhat  longer  than  the  remain- 
ing links  ;  again,  portions  of  the  chain  may  be  thinner 


270  BACTERIOLOGY. 

than  the  rest,  or  may  appear  broken  or  ragged.  Com- 
monly the  individuals  comprising  this  chain  of  cocci 
are  not  round,  but  appear  flattened  on  the  sides  adjacent 
to  one  another.  The  chains  are  sometimes  short,  con- 
sisting of  four  to  six  cells ;  or  again  they  may  be  much 
longer,  and  extend  from  a  half  to  two-thirds  across  the 
field  of  the  microscope. 

Under  artificial  conditions  it  sometimes  grows  well, 
and  can  be  cultivated  through  many  generations,  while 
at  other  times  it  rapidly  loses  its  vitality.  When  isolated 
from  the  diseased  area  upon  artificial  media  it  seems  to 
retain  its  vitality  for  a  longer  period  if  replanted  upon 
fresh  media  every  day  or  two  for  a  time ;  but  if  the 
first  generation  is  transplanted  and  is  allowed  to  re- 
main upon  the  original  medium,  it  is  not  uncommon 
to  find  the  organism  incapable  of  further  cultivation 
after  a  week  or  ten  days. 

Under  no  conditions  is  the  growth  of  this  organism 
very  luxuriant. 

On  gelatin  plates  its  colonies  appear  after  forty-eight 
to  seventy-two  hours  as  very  small,  flat,  round  points  of 
a  bluish-white  or  opalescent  appearance.  They  do  not 
cause  liquefaction  of  the  gelatin,  and  in  size  they  rarely 
exceed  0.6-0.8  mm.  in  diameter.  Under  low  magnify- 
ing power  they  have  a  brownish  or  yellowish  tinge  by 
transmitted  light,  and  are  finely  granular.  As  the  col- 
onies become  older  their  regular  border  may  become 
slightly  irregular  or  notched. 

In  stab-cultures  in  gelatin  they  grow  along  the  entire 
needle-track  as  a  finely  granular  line,  the  granules  rep- 
resenting minute  colonies  of  the  organism.  On  the 
surface  the  growth  does  not  usually  extend  beyond  the 
point  of  puncture. 


LESS  COMMON  PYOGENIC  ORGANISMS.        271 

On  agar-agar  plates  the  colonies  appear  as  minute 
pearly  points,  which  when  slightly  magnified  are  seen 
to  be  finely  granular,  of  a  light-brownish  color,  and 
regular  at  their  margins. 

When  smeared  upon  the  surface  of  agar-agar  or  gel- 
atin slants  the  growth  that  results  is  a  thin,  pearly, 
finely  granular  layer,  consisting  of  minute  colonies 
growing  closely  side  by  side.  Its  most  luxuriant 
growth  is  seen  on  glycerin-agar-agar  at  the  tempera- 
ture of  the  body  (37.5°  C.),  and  its  least  on  gelatin  at 
from  18°  to  20°  C. 

On  blood-serum  its  colonies  present  little  that  is  char- 
acteristic ;  they  appear  as  small,  moist,  whitish  points, 
from  0.6  to  0.8  mm.  in  diameter,  that  are  slightly  ele- 
vated above  the  surface  of  the  serum.  They  do  not 
coalesce  to  form  a  layer  over  the  surface,  but  remain  as 
isolated  colonies. 

On  potato  no  visible  development  appears,  but  after 
a  short  time  (thirty-six  to  seventy-two  hours)  there  is 
a  slight  increase  of  moisture  about  the  point  of  inocula- 
tion, and  microscopic  examination  shows  that  multiplica- 
tion of  the  organisms  placed  at  this  point  has  occurred. 

In  milk  its  conduct  is  not  always  the  same,  some  cult- 
ures causing  a  separation  of  the  milk  into  a  firm  clot 
and  colorless  whey,  while  others  do  not  produce  this 
coagulation.  The  latter,  when  cultivated  in  milk  of  a 
neutral  or  slightly  alkaline  reaction,  to  which  a  few 
drops  of  litmus  tincture  have  been  added,  produce  a 
very  faint  pink  color  after  twenty-four  hours  at  37.5° 
C. ;  there  is  no  coagulation. 

In  bouillon  it  grows  as  tangled  masses  or  clumps, 
which  upon  microscopic  examination  are  seen  to  consist 
of  long  chains  of  cocci  twisted  or  matted  together. 


272  BACTERIOLOGY. 

It  grows  best  at  the  temperature  of  the  body  (37.5° 
C.),  and  develops,  but  less  rapidly,  at  the  ordinary  room- 
temperature.  When  virulent  this  property  is  said  by 
Petruschky  to  be  preserved  by  retaining  the  cultures  in 
the  ice-chest  after  they  have  been  growing  on  gelatin 
for  two  days  at  22°  C. 

Its  virulence  may  often  be  increased  by  passing  it 
through  a  series  of  susceptible  animals. 

It  is  a  facultative  anaerobe. 

It  stains  with  the  ordinary  aniline  dyes,  and  is  not 
decolorized  when  subjected  to  Gram's  method. 

It  is  not  motile,  and,  being  a  micrococcus,  does  not 
form  endogenous  spores.  Under  artificial  conditions 
we  have  no  reason  to  believe  that  it  enters  a  stage  in 
which  its  resistance  to  detrimental  agencies  is  increased. 
In  the  tissues  of  the  body,  however,  it  appears  to  pos- 
sess marked  vitality,  for  it  is  not  rare  to  observe 
recurrences  of  inflammatory  conditions  due  to  this 
organism,  often  at  a  relatively  long  time  after  the 
primary  site  of  infection  has  healed. 

The  results  of  its  inoculation  into  the  tissues  of 
lower  animals  are  described  by  Rosenbach  and  Passet 
as  protracted,  progressive,  erysipelatoid  inflammations ; 
and  Fehleisen,  who  described  a  streptococcus  in  erysip- 
elas that  is  in  all  probability  identical  with  the  strepto- 
coccus pyogenes  under  consideration,  stated  that  it  pro- 
duced in  the  tissues  of  rabbits  (the  base  of  the  ear) 
a  sharply  defined,  migratory  reddening  without  pus- 
formation.  The  writer  has  encountered  a  culture  of 
this  organism  that  possessed  the  property  of  inducing 
erysipelas  when  introduced  into  the  skin  of  the  ear,  and 
disseminated  abscess-formation  when  injected  into  the 
circulation  of  rabbits.  This  observation  has  an  im- 


LESS  COMMON  PYOGENIC  ORGANISMS.        273 

portant  bearing  upon  the  question  concerning  the  iden- 
tity of  streptococci  found  in  various  inflammatory  con- 
ditions, such,  for  instance,  as  the  spreading  erysipelatoid 
manifestations  on  the  one  hand,  and  the  circumscribed 
abrscess-formations  on  the  other. 

The  results  that  follow  upon  the  inoculation  of  ani- 
mals with  cultures  of  streptococci  obtained  from  various 
inflammatory  lesions  are,  as  a  rule,  inconstant.  At 
times  cultures  will  be  encountered  that  are  apparently 
without  virulence,  no  matter  how  tested ;  while  again 
cultures  from  other  sources  exhibit  the  most  marked 
pathogenic  properties,  even  when  employed  in  almost 
infinitesimal  quantities.  Between  these  extremes  every 
gradation  may  be  expected.  The  virulence  of  a  culture 
is  not  necessarily  proportional  to  the  intensity  of  the 
pathological  process  from  which  it  is  derived. 

There  is  never  any  certainty  of  faithfully  repro- 
ducing, by  inoculation  into  susceptible  animals,  the 
pathological  lesion  from  which  a  culture  of  the  organ- 
ism may  have  been  obtained.  The  introduction  into  a 
susceptible  animal  of  a  culture  derived  from  either  a 
spreading  phlegmon  or  an  erysipelatous  inflammation 
may  result  in  erysipelas,  general  septicaemia,  local  ab- 
scess-formation, or,  as  said,  may  have  no  effect  at  all. 
Cultures  may  be  encountered  that  are  pathogenic  for 
one  susceptible  species  of  animals  and  not  for  another. 

Under  the  ordinary  conditions  of  artificial  cultiva- 
tion fully  virulent  varieties  of  streptococcus  pyogenes 
usually  lose  their  virulence  after  a  short  time. 
Petruschky1  preserves  this  property  by  cultivation 
upon  nutrient  gelatin  for  two  days  at  22°  C.,  keeping 

Petruschky:  Centralblatt  fur  Bakteriologie  und  Parasitenkunde, 
1895,  Abth.  i.  Band  xvii. 
18 


274  BACTERIOLOGY. 

the  cultures  after  this  time  in  the  refrigerator,  and 
transplanting  upon  fresh  gelatin  every  five  or  six  days. 
Marmorek1  finds  the  virulence  preserved  by  growing 
the  organism  in  a  mixture  of  2  parts  of  horse  or 
human  blood-serum  and  1  part  of  nutrient  bouillon,  or 
of  1  part  of  ascites-fluid  and  2  parts  of  bouillon. 

Certain  authors  are  of  the  opinion  that  a  relation 
exists  between  virulence  and  the  length  of  the  chains 
formed  by  streptococci  when  growing  in  fluid  media. 
It  is  held  that  those  forming  the  long  chains,  strepto- 
coccus longuSj  are  the  only  ones  concerned  in  animal 
pathology,  and  hence  the  only  ones  by  which  patho- 
genic powers  may  be  exhibited;  while  those  form- 
ing the  short  chains,  streptococcus  brevis,  are  not,  as  a 
rule,  pathogenic,  and  may  often  be  readily  differentiated 
from  the  other  variety  .by  more  or  less  gross  cultural 
characteristics,  such  as  slow  liquefaction  of  gelatin, 
visible  growth  on  potato,  etc.2 

ANTISTREPTOCOCCUS  SERUM. — For  a  time  a  great 
deal  of  interest  was  created  by  the  announcement  of 
Marmorek  that  he  had  succeeded  in  inducing  in  ani- 
mals a  state  of  immunity  from  streptococcus  infection, 
and  that  the  blood -serum  of  such  animals  when  injected 
into  other  susceptible  animals  and  human  beings  pos- 
sessed not  only  the  property  of  rendering  them  insus- 
ceptible to  this  particular  form  of  infection,  but  even 
exhibited  curative  powers  in  cases  already  infected. 
This  serum  was  obtained  from  horses  or  asses  that  had 
been  rendered  immune  by  the  gradual  introduction  into 

1  Marmorek :  Annales  de  Plnstitut  Pasteur,  1895. 

2  V.  Lingelsheim :  Zeitschrift  fiir  Hygiene,  1891,  Band  x.,  and  1892, 
Band  xii.     Behring:    Centralblatt  fur  Bakteriologie  imd  Parasiten- 
kunde,  1892,  Band  xii. 


LESS  COMMON  PYOGENIC  ORGANISMS.        275 

their  tissues  of  increasing  amounts  of  virulent  strepto- 
cocci. 

A  great  deal  has  been  said  both  for  and  against  the 
claims  of  Marmorek ;  but  at  present  the  bulk  of 
opinion  is  unfavorable,  or  at  least  very  conservative. 
Many  competent  clinicians  look  with  favor  upon  the 
use  of  this  serum  in  suitable  cases — i.  e.,  genuine,  un- 
complicated streptococcus  infections;  but  at  the  same 
time  others  have  failed  to  demonstrate  its  usefulness. 
Koch  and  Petruschky  failed  throughout  to  confirm 
Marmorek's  results.  It  is  difficult  to  reconcile  the  fact 
that  a  single  or  even  several  attacks  of  erysipelas  or  of 
phlegmonous  inflammation  in  the  human  being  afford 
no  protection  from  subsequent  attacks,  with  the  state- 
ment of  Marmorek  that  a  condition  of  complete  im- 
munity may  be  established  in  animals  by  the  repeated 
induction  of  mild  manifestations  of  the  disease. 

Streptococcus  pyogenes  is  the  organism  most  com- 
monly found  in  rapidly  spreading  suppuration,  while 
staphylococcus  pyogenes  aureus  is  most  frequently  found 
in  circumscribed  abscess-formations;  they  may  also  be 
found  together. 

NOTE. — If  the  opportunity  presents,  obtain  cultures 
from  a  case  of  erysipelas.  Compare  the  organism  thus 
obtained  with  the  streptococcus  just  mentioned.  Inoc- 
ulate rabbits  both  subcutaneously  and  into  the  circula- 
tion with  about  0.2  c.c.  of  pure  cultures  of  these  organ- 
isms in  bouillon.  Do  the  results  correspond,  and  do  they 
in  any  way  suggest  the  results  obtained  with  staphylo- 
coccns  pyogenes  aureus  when  introduced  into  animals  in 
the  same  way  ?  Do  these  streptococci  flourish  readily 
on  ordinary  media? 


276  BACTERIOLOGY. 

The  organisms  that  have  just  been  described  are 
commonly  known  as  the  "  pyogenic  cocci "  of  Ogston, 
Rosenbach,  and  Passet,  and  up  to  as  late  as  1885  were 
believed  to  be  the  specific  factors  concerned  in  the  pro- 
duction of  suppurative  inflammations.  Since  that  time, 
however,  there  has  been  considerable  modification  of 
this  view,  and  while  they  are  still  known  to  be  the 
most  common  causes  of  suppuration,  they  are  also 
known  not  to  be  the  only  causes  of  this  process. 

With  the  more  general  application  of  bacteriological 
methods  to  the  study  of  the  manifold  conditions  coming 
under  the  eye  of  the  physician,  the  surgeon,  and  the 
pathologist,  observations  are  constantly  being  made 
that  do  not  accord  with  the  earlier  ideas  upon  the 
specific  relation  of  the  pyogenic  cocci  to  all  forms  of 
suppuration.  There  is  an  abundance  of  evidence  to 
justify  the  opinion  that  a  number  of  organisms  not 
commonly  classed  as  pyogenic  may,  under  certain  cir- 
cumstances, assume  this  property.  For  example  : 

The  bacillus  of  typhoid  fever  has  been  found  in  pure 
culture  in  osteomyelitis  of  the  ribs,  in  acute  purulent 
otitis  media,  in  abscess  of  the  soft  parts,  in  the  pus 
of  empyema,  and  in  localized  nbro-peritonitis,  either 
during  its  course  or  as  a  sequel  of  typhoid  fever. 

Bacterium  coll  commune  has  been  found  in  pure  cult- 
ure in  acute  peritonitis,  in  liver-abscess,  in  purulent  in- 
flammation of  the  gall-bladder  and  ducts,  and  in  appen- 
dicitis; and  Welch1  has  found  it  in  pure  culture  in 
fifteen  different  inflammatory  conditions. 

MicrocoGcus  lanceolatus  (pneumococcus)  has  been 
found  alone  in  abscess  of  the  soft  parts,  in  purulent 

1  Welch :  "  Conditions  Underlying  the  Infection  of  Wounds,"  Ameri- 
can Journal  of  the  Medical  Sciences,  November,  1891. 


GONOCOCCUS.  277 

infiltration  of  the  tissues  about  a  fracture,  in  purulent 
cerebro-spinal  meningitis,  in  suppurative  synovitis,  in 
acute  pericarditis,  and  in  acute  inflammation  of  the 
middle  ear. 

Moreover,  many  of  the  less  common  organisms  have 
been  detected  in  pure  cultures  in  inflammatory  condi- 
tions with  which  they  were  not  previously  thought  to 
be  concerned,  and  to  which  they  are  not  usually  related 
etiologically. 

In  consideration  of  such  evidence  as  this  it  is  plain 
that  we  can  no  longer  adhere  rigidly  to  the  opinions 
formerly  held  upon  the  etiology  of  suppuration,  but 
must  subject  them  to  modifications  in  conformity  with 
this  newer  evidence.  We  now  know  that  there  exist 
bacteria  other  than  the  "  pyogenic  cocci/7  which,  though 
not  normally  pyogenic,  may  give  rise  to  tissue-changes 
indistinguishable  from  those  produced  by  the  ordinary 
pus-organisms.1 

GONOCOCCUS.      MICROCOCCUS   GONORRHCE.E. 

One  observes  upon  microscopic  examination  of  cover- 
slips  prepared  from  the  pus  of  acute  gonorrhrea  that 
many  of  the  pus-cells  contain  within  their  protoplasm 
numerous  small,  stained  bodies  that  are  usually  arranged 
in  pairs.  Occasionally  a  cell  is  seen  that  contains  only 
one  or  two  pairs  of  such  bodies ;  again,  a  cell  will  be 
encountered  that  is  packed  with  them.  Occasionally 
masses  of  these  small  bodies  will  be  seen  lying  free  in 
the  pus.  (See  Fig.  57.)  The  majority  of  the  pus-cells 
do  not  contain  them. 

1  Fora  more  detailed  discussion  of  the  subject,  see  "  The  Factors  Con- 
cerned in  the  Production  of  Suppuration,"  International  Medical 
Magazine,  Philadelphia,  May,  1892. 


278  BACTERIOLOGY. 

These  small,  round,  or  oval  bodies  are  the  so-called 
"gonococci"  discovered  by  Neisser,  and  more  fully 
studied  subsequently  by  Bumm,  to  whom  we  are  in- 
debted for  much  of  our  knowledge  concerning  them. 

As  the  name  implies,  this  organism  is  a  micrococcus, 


FIG.  57. 


- 

Pus  of  gonorrhoea,  showing  diplococci  in  the  bodies  of  the  pus-cells. 

and  as  it  is  commonly  arranged  in  pairs  (flattened  at 
the  surfaces  in  juxtaposition)  it  is  often  designated  as 
diplococcus  of  gonorrhoea.  It  is  always  to  be  found  in 
gonorrhceal  pus,  and  often  persists  in  the  urethral  dis- 
charges and  secretions  far  into  the  stage  of  conva- 
lescence. It  is  not  present  in  inflammatory  conditions 
other  than  those  of  gonorrhoeal  origin. 

It  is  easily  detected  microscopically  in  the  secretions 
of  acute  gonorrhoea.  In  secondary  lesions  and  in  very 
old,  chronic  cases  it  is  difficult  of  detection  and  fre- 
quently eludes  all  efforts  to  find  it.  It  is  stained  by  the 
ordinary  methods,  but  perhaps  most  satisfactorily  with 
the  alkaline  solution  of  methylene-blue.  Most  impor- 
tant as  a  differential  test  is  its  failure  to  stain  by  the 
method  of  Gram.  (How  does  this  compare  with  the 


GONOCOCCUS.  279 

behavior  of  the  other  pyogenic  cocci  when  treated  in 
the  same  way  ?) 

It  does  not  grow  upon  ordinary  nutrient  media, 
and  has  only  been  isolated  in  culture  through  the  em- 
ployment of  special  methods.  Its  growth  under  arti- 
ficial conditions  seems  to  depend  upon  some  partic- 
ular nutrient  substance  that  is  supplied  by  blood  or 
blood-serum,  and  in  all  the  media  that  have  been  suc- 
cessfully used  for  its  cultivation  this  substance  is 
apparently  an  essential  constituent.  By  many  investi- 
gators it  is  thought  that  only  human  blood  possesses 
this  important  ingredient,  though  this  opinion  is  not 
universal.1 

It  was  first  isolated  in  culture  by  Bumm,  who  used 
for  this  purpose  coagulated  human  blood-serum  ob- 
tained from  the  placenta. 

Wertheim  improved  the  method  of  Bumm  by  using 
a  mixture  of  equal  parts  of  sterile  human  blood-serum 
and  ordinary  sterilized  nutrient  agar-agar,  the  latler 
having  been  liquefied  and  kept  at  50°  C.  until  after 
the  mixture  was  made,  when  it  was  allowed  to  cool  and 
solidify. 

Other  investigators  have  substituted  for  human  blood- 
serum  certain  pathological  fluids  from  the  human  body, 
such  as  ascites-fluid,  fluid  from  ovarian  cysts,  and  serous 
effusions  from  the  pleura  and  from  the  joint-cavities. 

The  method  used  by  Pfeiffer  for  the  cultivation  of 
the  bacillus  of  influenza  is  also  said  to  have  been  suc- 
cessfully employed.  Abel  recommends  a  needle-prick 
in  the  finger  as  a  most  convenient  source  from  which  to 

1  An  instructive  article  on  this  subject  is  that  by  Foulerton :  "  On 
Micrococcus  Gonorrhoeas  and  Gonorrhceal  Infection,"  Transactions  of 
the  Bristol  Institute  of  Preventive  Medicine,  1897,  series  i 


280  BACTERIOLOGY. 

obtain  the  necessary  amount  of  human  blood  that  is  to 
be  smeared  over  the  surface  of  the  slanting  agar-agar 
when  Pfeiffer's  method  is  employed. 

"Wright's  modification  of  Steinschneider's  method  has 
given  such  satisfactory  results  in  his  hands  that  it  will 
be  given  here  somewhat  in  detail.  The  medium  con- 
sists of  a  mixture  of  urine,  blood-serum  (human  or 
bovine,  either  serving  the  purpose),  and  nutrient  agar- 
agar.  The  urine  and  blood-serum  are  collected  with- 
out special  aseptic  precautions,  and  subsequently  freed 
from  bacteria  by  filtration  through  unglazed  porcelain. 
Frequently  this  is  the  tedious  part  of  the  process,  as 
the  serum  and  urine  pass  very  slowly  through  the 
porcelain  filters  generally  employed  in  laboratories. 
Wright  recommends  a  filtering  cylinder  manufactured 
by  the  Boston  Filter  Company  as  an  apparatus  that  not 
only  gives  a  sterile  filtrate,  but  also  permits  of  very 
rapid  passage  of  the  fluid. 

The  details  of  the  method  as  given  by  Wright  are  as 
follows :  "  A  litre  of  nutrient  agar  is  prepared  in  the 
usual  manner,  and  after  filtration  it  is  evaporated  to 
about  600  c.c.  This  concentration  is  desirable,  so  that 
after  dilution  with  the  urine  and  serum  the  medium 
may  be  sufficiently  firm.  This  concentrated  agar  is  then 
run  into  test-tubes  and  the  whole  sterilized  by  steam  on 
three  successive  days.  The  quantity  of  agar  placed  in 
each  tube  is  smaller  than  is  usual ;  this  is  in  order  to 
allow  for  the  subsequent  addition  of  the  urine  and 
serum. 

"  The  blood-serum,  which  need  not  be  free  from  cor- 
puscles, is  first  passed  through  white  sand,  which  is 
supported  in  a  funnel  by  filter-paper,  in  order  to  re- 
move as  far  as  is  possible  any  particles  in  suspension, 


GONOCOCCUS.  281 

and  is  then  mixed  with  half  its  volume  of  fresh  urine. 
The  mixture  of  urine  and  blood-serum  is  next  filtered 
by  suction  through  an  unglazed  porcelain  cylinder  into 
a  receiving-flask,  such  as  chemists  use  for  similar  pur- 
poses, by  means  of  a  water-vacuum  pump.  This  frees 
the  mixture  from  bacteria. 

"  The  usual  precautions  are,  of  course,  taken  to  pre- 
vent the  contamination  of  the  filtrate,  such  as  the  pre- 
vious sterilization  by  steam  of  the  cylinder  and  receiv- 
ing-flask, besides  others  which  will  occur  to  any  bacteri- 
ologist. 

"  To  the  agar  in  each  test-tube,  which  is  fluid  and  of 
a  temperature  of  about  40°  C.,  there  is  added  about 
one-third  to  one-half  its  volume  of  the  filtered  mixture 
of  urine  and  blood-serum.  This  is  conveniently  accom- 
plished by  pouring  the  mixture  from  the  receiving-flask 
through  the  lateral  tube,  inserted  near  its  neck  directly 
into  the  tubes.  The  preliminary  melting  of  the  agar 
is  best  eifected  in  the  steam  sterilizer,  in  order  that  any 
organisms  which  have  found  lodgement  in  the  cotton 
plugs  of  the  tubes  may  be  destroyed.  When  the  agar 
is  melted  it  is  cooled  and  kept  fluid  by  placing  the 
tubes  in  a  water-bath  at  40°  C.  Each  tube,  after  the 
addition  of  the  urine  and  serum  to  the  fluid  agar,  is 
quickly  shaken  to  insure  a  uniform  mixture,  and  is 
then  placed  in  an  inclined  position  to  allow  the  agar  to 
solidify  with  a  slanting  surface.  When  the  medium 
in  the  tubes  has  solidified  the  tubes  are  placed  in  the 
incubator  for  about  twenty-four  hours  to  test  for  con- 
taminations, after  which  they  are  ready  for  use." 

The  successive  dilutions  are  now  to  be  made  upon 
the  slanting  surface  of  this  mixture,  as  the  mass  in  the 
tubes  cannot  be  redissolved  without  exposure  to  a  de- 


282  BACTERIOLOGY. 

gree  of  heat  that  apparently  interferes  with  the  nutri- 
tive value  of  the  serum  contained  in  the  medium. 

When  inoculated  with  gonorrhreal  pus,  by  smearing 
a  loopful  over  the  surface,  the  tubes  are  to  be  kept  at 
from  37°  to  38°  C.  The  organism  does  not  develop 
properly  at  a  temperature  below  this  point. 

After  twenty-four  hours  the  colonies  of  the  gono- 
coccus  appear  on  the  surface  of  the  medium,  accord- 
ing to  Wright,  as  very  tiny,  grayish,  semi-translucent 
points.  After  forty-eight  hours  they  may  be  about 
1  millimetre  or  so  in  diameter,  slightly  elevated,  with 
a  rounded  outline,  grayish  in  color,  and  semi-translu- 
cent by  transmitted  light.  By  reflected  light  their  sur- 
face has  the  appearance  of  frosted  glass.  Later,  if  few 
in  number,  so  that  their  growth  is  unimpeded,  the  colo- 
nies may  attain  a  diameter  of  2  millimetres  or  more,  be- 
come thicker  and  denser,  with  a  faintly  brownish  tinge 
about  their  centres,  and  a  slightly  irregular  outline. 

Under  a  low  power  of  the  microscope  a  fully  de- 
veloped colony  is  seen  to  consist  of  a  general  circular 
expansion,  with  thin,  translucent,  smooth,  sharply  de- 
fined margin,  but  becoming  brownish,  granular,  and 
thicker  toward  the  central  portion,  which  is  made  up 
of  coarse,  granular,  brown-colored  clumps  closely  packed 
together. 

The  appearances  coincide  with  the  figure  of  such  a 
colony  given  by  Wertheim.1 

Wassermann  2  calls  attention  to  the  success  he  has 
had  in  cultivating  this  organism  upon  a  mixture  of 
swTine-serum  and  nitrose,  the  latter  being  a  com- 

1  Deutsche  med.  Wochenschrift,  1891,  No.  50  ;  Centralblatt  fiir  Gyna- 
kologie,  1891,  No.  24. 

2  Zeitschrift  fiir  Hygiene  und  Infektiouskrankheiten,  Bd.  xvii.  p.  298. 


GONOCOCCUS.  283 

mercial  product  chemically  known  as  casein-sodium 
phosphate. 

The  preparation  of  the  medium  and  its  composition 
arc  as  follows  : 

In  an  Erlenmeyer  flask  mix  15  c.c.  of  swine-serum,  as 
free  as  possible  from  haemoglobin ;  30  to  35  c.c.  of  water ; 
2  to  3  c.c.  of  glycerin ;  and  finally  0.8  to  0.9  gramme 
(/.  e.,  about  2  per  cent.)  of  nitrose.  This  is  boiled,  with 
gentle  agitation,  over  a  free  flame,  until  all  ingredients 
are  dissolved  and  the  cloudy  fluid  has  become  quite 
clear.  After  such  boiling  the  mixture  can  be  sterilized 
by  steam  without  precipitating  the  albumin,  and  may 
then  be  kept  indefinitely  ready  for  use. 

When  needed,  the  flask  and  its  contents  are  heated  to 
50°  C. ;  from  six  to  eight  tubes  of  2  per  cent,  peptone- 
agar-agar  are  dissolved  by  boiling,  brought  to  50°  C., 
and  then  mixed  with  the  solution  in  the  flask  and  the 
mass  poured  into  Petri  dishes.  Upon  the  surface  of  this 
serum-nitrose-agar  the  cultivation  is  to  be  conducted. 
\Vn<sermann  lays  particular  stress  upon  two  points  that 
are  essential  to  success,  viz.,  the  preliminary  boiling  of 
the  serum-nitrose  mixture  before  steam  sterilization,  as 
this  prevents  precipitation  of  the  albumin ;  and  the 
necessity  of  having  both  the  serum-nitrose  mixture  and 
the  agar-agar,  to  be  mixed  with  it,  at  not  over  50°  C., 
for  if  they  are  at  boiling  temperature  when  mixed,  or  if 
they  are  brought  to  the  boiling  temperature  after  mixing, 
the  albumin  will  be  precipitated  notwithstanding  the 
presence  of  the  nitrose,  which  otherwise  prevents  this. 

Wassermann  further  observes  that  some  samples  of 
serum  require  to  be  more  highly  diluted  with  water  than 
in  the  proportions  given  above ;  that  the  agar-agar 
should  be  feebly,  but  distinctly,  alkaline  to  litmus, 


284  BACTERIOLOGY. 

causing  no  reddening  whatever  of  blue  litmus  paper ; 
and,  finally,  that  the  Petri  dishes  containing  the  solidi- 
fied medium  on  which  the  cultures  are  growing  are  best 
kept  bottom  upward,  so  as  to  prevent  water  of  con- 
densation collecting  on  the  surface.  By  the  use  of  the 
above  medium  he  has  cultivated  the  gonococcus  from 
about  one  hundred  different  cases. 

If  transplanted  from  the  original  culture  to  either 
glycerin-agar-agar  or  to  Loffler's  serum-mixture,  a 
growth  is  sometimes  observed,  more  often  in  the  latter 
than  in  the  former,  but  of  so  feeble  a  nature  that  these 
substances  cannot  be  regarded  as  suitable  for  its  culti- 
vation. As  a  rule,  development  does  not  occur  on 
glycerin-agar. 

Microscopic  examination  of  colonies  of  this  organism 
reveals  the  presence  of  a  diplococcus  somewhat  larger 
than  the  ordinary  pyogenic  cocci.  The  opposed  sur- 
faces of  the  individual  cells  that  comprise  the  couplets 
are  flattened  and  separated  by  a  narrow  slit.  At  times 
the  cocci  are  arranged  as  tetrads. 

This  organism  cannot  be  grown  at  a  temperature 
lower  than  that  of  the  human  body,  and  cultures  that 
have  been  obtained  by  either  of  the  favorable  methods 
are  said  to  lose  their  vitality  when  kept  at  ordinary 
room-temperature  for  about  two  days. 

It  is  killed  in  a  few  hours  by  drying. 

Cultures  retain  their  vitality  under  favorable  condi- 
tions of  nutrition,  temperature,  and  moisture  for  from 
three  to  four  weeks. 

This  organism  is  without  pathogenic  properties  for 
monkeys,  dogs,  and  horses,  as  well  as  for  the  ordinary 
smaller  animals  used  for  this  purpose  in  the  laboratory. 


GONOCOCCUS.  285 

In  man  typical  gonorrhoea  has  been  produced  on 
several  occasions  by  the  introduction  into  the  urethra 
of  pure  cultures  of  this  organism. 

In  addition  to  its  causal  relation  to  specific  ure- 
thritis,  it  is  the  cause  of  gonorrhceal  prostatitis  in 
man,  of  gonorrhoeal  proctitis  in  both  sexes,  and  of  gon- 
orrhceal inflammation  of  the  urethra,  of  Bartholin's 
glands,  of  the  cervix  uteri,  and  of  the  vagina  in 
women  and  young  girls.  It  is  etiologically  related  to  the 
specific  conjunctivitis  (ophthalmia  neonatorum)  of  young 
infants,  and  also  occasionally  to  ophthalmia  in  adults. 

Secondarily,  it  is  concerned  in  specific  inflammations 
of  the  tubes  and  ovaries,  of  the  lymphatics  communi- 
cating with  the  genitalia,  of  the  serous  surfaces  of  joints, 
and  of  those  of  the  heart,  lungs,  and  abdominal  cavity. 

Other  species  of  micrococci  have  from  time  to  time 
been  described  as  occurring  in  the  pus  of  acute  urethritis 
and  of  other  purulent  inflammations.  Many  of  these 
are  of  no  significance.  Some  of  them  possess  peculiarities 
that  might  lead  to  confusion.  The  diplococcus  described 
by  Heiman1  has  certain  points  of  resemblance  to  the 
gonococcus,  such  as  its  location  in  the  bodies  of  pus- 
cells,  its  grouping  as  diplococci,  its  size  and  general  ap- 
pearance ;  but  it  is  still  readily  distinguished  from  the 
gonococcus  by  its  retention  of  color  when  stained  by 
Gram's  method.  The  diplococcus  detected  by  Bumm 
in  puerperal  cystitis  is  likewise  often  found  within  pus- 
cells;  but  it  is  readily  differentiated  from  the  gono- 
coccus by  its  growth  upon  ordinary  nutrient  media. 
Diplococcus  intracellularis  meningitidis  of  Weichsel- 
baum,  isolated  from  the  pus  of  cerebro-spinal  menin- 
gitis, is  microscopically  also  strikingly  like  the  gono- 

1  New  York  Medical  Record,  June  22,  1895. 


28  6  B A  CTERIOLOG  Y. 

coccus  as  it  is  seen  in  pus;  but,  unlike  the  latter 
organism,  may  easily  be  cultivated  by  the  ordinary 
culture-methods. 

POSITIVE  AND  NEGATIVE  DISTINGUISHING  PECULI- 
ARITIES OF  THE  GONOCOCCUS. — Since  gonorrho3al  dis- 
charges may  be  contaminated  with  pyogenic  cocci  other 
than  those  causing  the  specific  inflammation,  it  is  im- 
portant in  efforts  to  isolate  this  organism  that  the  dif- 
ferential tests  be  borne  in  mind  and  put  into  practice. 
The  gonococcus  is  differentiated  from  the  commoner 
pyogenic  organisms  by  the  following  peculiarities : 

First,  it  is  practically  always  seen  in  the  form  of  dip- 
lococci,  the  pair  of  individual  cells  having  the  appear- 
ance of  two  hemispheres,  with  the  diameters  opposed 
and  separated  from  one  another  by  a  narrow,  colorless 
slit.  (Is  this  the  case  with  staphylococcus  or  strepto- 
coccus pyogenes  f) 

Second,  in  gonorrhoaal  pus  it  is  practically  always  to 
be  found  within  the  protoplasmic  bodies  of  pus-cells. 
(How  does  this  compare  with  the  conditions  found  in 
ordinary  pus  ?) 

Third,  it  stains  readily  with  the  ordinary  staining- 
reagents,  but  loses  its  color  when  treated  by  the  method  of 
Gram.  (Treat  a  cover-slip  from  ordinary  pus  by  this 
method  and  note  the  result.) 

Fourth,  it  does  not  develop  upon  any  of  the  ordinary 
media  used  in  the  laboratory ;  while  the  common  pus- 
organisms,  with  perhaps  the  exception  of  the  strepto- 
cocci, are  vigorous  growers  and  are  not  markedly  fas- 
tidious as  to  their  nutritive  medium. 

Fifth,  when  obtained  in  pure  culture  by  either  of  the 
special  procedures  noted  above,  its  cultivation  may  be 
continued  upon  the  same  medium ;  but  growth  will 


BACILLUS  PYOCYANEUS.  287 

usually  not  be  observed  if  it  is  transplanted  to  ordi- 
nary nutrient  gelatin,  agar-agar,  bouillon,  or  potato ; 
should  it  grow  under  these  circumstances  its  develop- 
ment will  be  very  feeble.  (Is  this  the  case  with  com- 
mon pus-producers?) 

Sixth,  it  has  no  pathogenic  properties  for  animals, 
while  several  of  the  pyogenic  cocci,  notably  staphylo- 
coccus  aureus  and  streptococcus  pyogenes,  are  usually 
capable  of  exciting  pathological  conditions.  (This  is 
less  commonly  true  of  streptococcus  pyogenes  than  of 
staphylococcus  aureus.} 

BACILLUS    PYOCYANEUS  (BACILLUS   OF   GREEN   PUS). 

Another  common  organism  that  may  properly  be 
mentioned  at  this  place,  though  perhaps  not  strictly 
pyogenic,  is  a  bacillus  frequently  found  in  discharges 
from  wounds,  viz.,  bacillus  pyocyaneus,  or  bacillus  of 
green  pus,  or  of  blue  pus,  or  of  blue-green  pus,  as  it 
is  commonly  called.  Bacillus  pyocyaneus  is  a  deli- 
cate rod  with  rounded  or  pointed  ends.  It  is  actively 
motile  ;  does  not  form  spores.  As  seen  in  preparations 
made  from  cultures  it  is  commonly  clustered  in  irreg- 
ular masses.  It  does  not  form  long  filaments,  there 
being  rarely  more  than  four  joined  end  to  end,  and 
most  frequently  occurs  as  single  cells. 

It  grows  readily  on  all  artificial  media,  and  gives  to 
some  of  them  a  bright-green  color  that  is  most  conspic- 
uous where  it  is  in  contact  with  the  air.  This  green 
color,  which  becomes  more  and  more  marked  as  growtli 
advances,  is  not  seen  in  the  growtli  itself  to  any  extent, 
but  is  diffused  through  the  medium  on  which  the  organ- 
ism is  developing.  Ultimately  this  color  becomes  much 
darker,  and  in  very  old  agar-agar  cultures  may  become 


288 


BACTERIOLOGY. 


almost  black  (sometimes  very  dark-blue  green,  at  others 
brownish-black). 

Its  growth  on  gelatin  in  stab-cultures  is  accompanied 
by  liquefaction  and  the  diffusion  of  a  bright-green  color 
throughout  the  surrounding  unliquefied  medium.  As 
liquefaction  continues,  and  the  whole  of  the  gelatin 
ultimately  becomes  fluid,  the  green  color  is  confined 


FIG.  58. 


FIG.  59. 


Colony  of  b.  pyocyaneus  after  twenty-four  hours 
on  gelatin  at  20°-22°  C. 


FIG.  60. 


Stab-culture  of  6. 
pyocyaneus  in  gel- 
atin after  twenty- 
eight  hours  at  22°C. 


Colony  of  6.  pyocyaneus  after  forty-two  hours  on 
gelatin  at  20°-22°  C. 


to  the  superficial  layers  in  contact  with  the  air.  The 
form  taken  by  the  liquefying  portion  of  the  gelatin  in 
the  earliest  stages  of  development  is  somewhat  that  of 
an  irregular,  slender  funnel.  (See  Fig.  58.) 

On  gelatin  plates  the  colonies  develop  rapidly ;  they 


BACILLUS  PYOCYANEUS.  289 

are  not  sharply  circumscribed,  but  usually  present  at 
ti  r.-t  a  fringe  of  delicate  filaments  about  their  periphery. 
(See  Fig.  59.)  As  growth  progresses  and  liquefaction 
becomes  more  advanced  the  central  mass  of  the  colony 
sinks  into  the  liquid,  while  at  the  same  time  there  is 
an  extension  of  the  colony  laterally.  At  this  stage  the 
colony,  when  slightly  magnified,  may  present  various 
appearances,  the  most  common  being  that  shown  in 
Fig.  60. 

The  gelatin  between  the  growing  colonies  takes  on  a 
bright  yellowish-green  color ;  but  as  growTth  is  compar- 
atively rapid,  it  is  quickly  entirely  liquefied,  and  one 
often  sees  the  colonies  floating  about  in  the  pale-green 
fluid. 

On  agar-agar  the  growth  is  dry,  sometimes  with  a 
slight  metallic  lustre,  and  is  of  a  whitish  or  greenish- 
white  color,  while  the  surrounding  agar-agar  is  bright 
green.  With  time  this  bright  green  becomes  darker, 
pacing  into  blue-green,  and  finally  turns  almost  black. 

On  potato  the  growth  is  brownish,  dry,  and  slightly 
elevated  above  the  surface.  In  some  cultures  the 
potato  about  the  line  of  growth  becomes  green;  in 
others  this  change  is  not  so  noticeable.  With  many 
cultures  a  peculiar  phenomenon,  consisting  of  a  change 
of  color  from  brown  to  green,  may  be  produced  by 
lightly  touching  the  growth  with  a  sterile  platinum 
needle.  The  change  occurs  only  at  the  point  touched. 
It  is  best  seen  in  cultures  that  have  been  kept  in  the 
incubator  for  from  seventy-two  to  ninety-six  hours.  It 
occurs  in  from  one  to  three  minutes  after  touching  with 
the  needle,  and  may  last  from  ten  minutes  to  half  an 
hour.  This  is  the  "chameleon  phenomenon"  of  Paul 
Ernst. 

19 


290  BACTERIOLOGY. 

In  bouillon  the  green  color  appears,  and  the  growth 
is  seen  in  the  form  of  delicate  flocculi.  A  very  delicate 
mycoderma  is  also  produced. 

In  milk  it  causes  an  acid  reaction,  with  coincident 
coagulation  of  the  casein. 

On  blood-serum  and  egg-albumin  its  growth  is  ac- 
companied by  liquefaction.  The  growth  on  coagulated 
egg-albumin  is  seen  as  a  dirty-gray  deposit  surrounded 
by  a  narrow  brownish  zone ;  the  remaining  portion  of 
the  medium  is  bright  green  in  color.  As  the  culture 
becomes  older  the  green  may  give  way  to  a  brown  dis- 
coloration. 

In  peptone  solution  (double  strength)  it  causes  a 
bluish-green  color.  In  one  of  four  cultures  from  differ- 
ent sources  we  observed  the  production  of  a  distinct 
blue  color. 

It  produces  indol. 

It  stains  with  the  ordinary  dyes,  and  its  flagella 
may  be  readily  demonstrated  by  Loffler's  method  of 
staining. 

INOCULATION  INTO  ANIMALS. — As  a  rule,  cultures 
of  this  organism  obtained  directly  from  the  discharges 
of  the  wound  are  capable,  when  introduced  into  ani- 
mals, of  producing  diseased  conditions ;  but  cultures 
kept  on  artificial  media  for  a  long  time  may  in  part,  or 
completely,  lose  this  power. 

When  guinea-pigs  or  rabbits  are  inoculated  subcuta- 
neously  with  1  c.c.  of  virulent  fluid  cultures  of  this 
organism,  death  usually  results  in  from  eighteen  to 
thirty-six  hours.  At  the  seat  of  inoculation  there  are 
found  an  extensive  purulent  infiltration  of  the  tissues 
and  a  marked  zone  of  inflammatory  redema. 

When  introduced  directly  into  the  peritoneal  cavity 


BACILLUS  PYOCYANEUS.  291 

the  results  are  also  fatal,  and  at  autopsy  a  genuine 
fibrinous  peritonitis  is  found.  There  is  usually  an  ac- 
cumulation of  serum  in  both  the  peritoneal  and  pleural 
cavities.  At  autopsies  after  both  methods  of  inocula- 
tion the  organisms  will  be  found  in  pure  cultures  in 
the  blood  and  internal  viscera. 

When  animals  are  inoculated  with  small  doses  (less 
than  1  c.c.  of  a  bouillon  culture)  of  this  organism  death 
may  not  ensue,  and  only  a  local  inflammatory  reaction 
(abscess-formation)  may  be  set  up.  In  these  cases  the 
animals  are  usually  protected  against  subsequent  inocu- 
lation with  doses  that  would  otherwise  prove  fatal. 

Most  interesting  in  connection  with  bacillus  pyocy- 
aneus  is  the  fact,  as  brought  out  in  the  experiments  of 
Bouchard,  and  of  Charrin  and  others,  that  its  products 
possess  the  power  of  counteracting  the  pathogenic  ac- 
tivities of  bacillus  anthrads.  That  is  to  say,  if  an" 
animal  be  inoculated  with  a  virulent  anthrax  culture, 
and  soon  after  be  inoculated  with  a  culture  of  bacillus 
pyocyaneuSj  the  fatal  effects  of  the  former  inoculation 
may  be  prevented.  Emmerich  and  Low  *  are  inclined 
to  attribute  this  to  the  direct  bacteriolytic  action  of  the 
pyocyaneus  enzymes  upon  the  anthrax  bacilli  introduced 
into  the  tissues. 

In  the  literature  upon  the  green-producing  organisms 
that  have  been  found  in  inflammatory  conditions  sev- 
eral varieties — believed  to  be  distinct  species — have 
been  described ;  but  when  cultivated  side  by  side  their 
biological  differences  are  seen  to  be  so  slight  as  to  ren- 
der it  probable  that  they  are  but  modifications  of  one 
and  the  same  species. 

1  Munchener  med.  Wochenschrift,   1898,  No.  40 ;  Centralblatt  fur 
Bakteriologie  und  Parasitenkunde,  1899,  Abt.  i.  No.  1,  p.  33. 


292  BACTERIOLOGY. 


THE   BACILLUS   OF  BUBONIC   PLAGUE. 

Before  passing  from  the  subject  of  suppuration  it 
may  not  be  inappropriate  to  call  attention  to  the  light 
that  modern  methods  of  investigation  have  shed  upon 
the  etiology  of  bubonic  plague,  an  epidemic  disease 
characterized  by  "suppuration  of  the  lymphatic  glands, 
~and  accoTfipanied  By  a  veryTngff  fate  ~6f  mortality. 

This  pestilence,  probably  endemic  in  certain  sections 
of  the  Orient,  is  one  of  the  most  conspicuous  epidemic 
diseases  of  history.  Since  early  in  the  Christian  era  epi- 
demics and  pandemics  of  plague  have  made  their  appear- 
ance in  Europe  at  different  times.  During  and  for  a  time 
after  the  Middle  Ages  it  was  more  or  less  frequent  in 
India,  China,  Arabia,  Northern  Africa,  Italy,  France, 
Germany,  and  Great  Britain.  In  history  it  is  variously 
known  as  the  "  Justinian  Plague  "  of  the  sixth  century, 
the  "  Black  Death  "  of  the  fourteenth  century,  and  the 
"  Great  Plague  of  London  "  of  the  seventeenth  century, 
though  it  is  difficult  to  say  to  what  extent  these  pesti- 
lences were  uncomplicated  manifestations  of  genuine 
bubonic  plague.  During  the  existence  of  the  Justinian 
Plague  10,000  people  are  said  to  have  died  in  Con- 
stantinople in  a  single  day,  and  Hecker  estimates  that 
during  the  pandemic  of  the  Black  Death  25,000,000 
people  (a  quarter  of  the  entire  population  of  Europe) 
succumbed  to  the  disease.  During  the  Great  Plague 
of  London  (1664-'65)  the  total  mortality  for  one  year 
was  68,596,  out  of  an  estimated  population  of  460,000 
souls. 

It  is  not  surprising  to  learn  that  it  was  to  guard 
against  the  plague  that  quarantine  regulations  were 
first  established. 


THE  BACILLUS  OF  BUBONIC  PLAGUE.        293 

The  first  and  certainly  the  most  exact  information 
up  to  date  concerning  the  cause  and  pathology  of  the 
plague  resulted  from  the  investigations  of  Yersin,  of 
Kitusato,  and  of  Aoyama,  conducted  during  the  epi- 
demic of  1894  in  Hong-Kong,  China;  though  since 
then  numerous  other  investigators  have  made  addi- 

FIG.  61. 
A 


Bacillus  of  bubonic  plague :  A,  in  pus  from  suppurating  bubo ;  B,  the 
bacillus  very  much  enlarged  to  show  peculiar  polar  staining. 

tional  important  contributions  to  our  knowledge  of 
the  subject.  The  results  of  these  studies  indicate  that 
bubonic  plague  is  an  infectious,  not  markedly  con- 
tagious, disease  that  depends  for  its  existence  upon  the 
presence  in  the  tissues  of  a  specific  micro-organism — 
the  so-called  plague  or  pest  bacillus. 


294  BACTERIOLOGY. 

This  organism  is  described  as  a  short,  oval  bacillus, 
usually  seen  single,  sometimes  joined  end  to  end  in  pairs 
or  threes,  less  commonly  as  longer  threads.  It  stains 
more  readily  at  its  ends  than  at  its  centre.  It  is  some- 
times capsulated ;  is  non-spore-forming ;  is  aerobic,  and 
is  non-motile.  It  is  found  in  large  numbers  in  suppu- 
rating glands,  and  in  much  smaller  numbers  in  the  cir- 
culating blood.  (Fig.  61.) 

It  is  demonstrable  in  cover-slip  preparations  made 
from  the  pus  and  in  sections  of  the  glands  by  the  ordi- 
nary staining-methods. 

Wilm  l  has  found  it  by  culture  methods  in  the  spleen, 
lungs,  liver,  kidneys,  stomach,  walls  of  the  intestine, 
urine,  and  intestinal  contents  of  fresh  cadavers ;  and 
during  life  in  the  blood,  expectoration,  faBces,  and  urine 
of  patients  sick  of  plague.  He  failed  to  find  it  in  the 
perspiration. 

Yersin  states  that  it  does  not  retain  the  color  when 
treated  by  the  method  of  Gram ;  while  Kitasato  says 
that  it  at  one  time  stains  by  this  method  and  at  another 
it  becomes  decolorized.  Aoyama  observed  that  those 
bacilli  within  the  suppurating  glands  were  decolorized, 
while  those  in  the  blood  retained  the  stain  when  treated 
by  Gram's  method. 

Since  there  is  often  a  mixed  infection  in  these  cases, 
it  appears  likely  that  the  above  discrepancy  may  be 
attributed  to  individual  peculiarities  of  different  species 
of  bacteria  that  were  under  examination,  an  opinion 
that  is  borne  out  by  more  recent  studies,  from  which  it 
has  been  decided  that  the  genuine  plague  or  pest-bacillus 
does  not  stain  by  Gram's  method. 

It  may  be  cultivated  upon  ordinary  nutrient  media, 
1  Wilm :  Hygienische  Kundschau,  1897,  p.  217. 


THE  BACILLUS  OF  BUBONIC  PLAGUE.        295 

though  preference  is  given  to  a  neutral  or  slightly 
alkaline  '1  per  cent,  peptone  solution  containing  from 
1  to  2  per  cent,  of  gelatin. 

The  most  favorable  temperature  for  its  growth  is 
between  36°  and  39°  C.  Its  colonies  on  glycerin-agar- 
agar  and  on  coagulated  blood-serum  are  described  as 
iridescent,  transparent,  and  whitish.  On  gelatin  at 
18°-20°  C.  it  develops  as  small,  sharply  defined,  white 
colonies  without  liquefaction  of  the  medium.  In  stab- 
cultures  it  develops  both  on  the  surface  and  along  the 
track  of  the  needle.  Its  growth  is  slow.  It  does  not 
cause  a  diffuse  clouding  of  bouillon,  but  grows  rather 
as  irregular,  flocculent  clumps  that  adhere  to  the  sides 
or  sink  to  the  bottom  of  the  vessel,  leaving  the  fluid 
clear.  It  shows  but  limited  growth  on  potato.  It  does 
not  ferment  glucose  with  production  of  gas,  nor  does  it 
form  indol.  It  coagulates  milk. 

This  organism  is  killed  by  drying  at  ordinary  room- 
temperature  in  four  days.  It  is  killed  in  three  to  four 
hours  by  direct  sunlight.  It  is  destroyed  in  a  half  hour 
by  80°  C.,  and  in  a  few  minutes  by  100°  C.  (steam). 
It  is  killed  in  one  hour  by  1  per  cent,  carbolic  acid 
and  in  two  hours  by  1  per  cent,  milk  of  lime. 

It  is  pathogenic  for  rats,  mice,  guinea-pigs,  rabbits, 
hogs,  horses,  monkeys,  cats,  chickens,  and  sparrows. 
Pigeons,  hedgehogs,  and  frogs  are  immune,  and  dogs 
and  bovines  are  apparently  so.1  Animals  succumb  to 
subcutaneous  inoculation  in  from  two  to  three. days. 
According  to  Yersin,  the  site  of  subcutaneous  inocu- 
lation becomes  redematous  and  the  neighboring  lym- 
phatics are  enlarged  in  a  few  hours.  After  twenty-four 

1  Nuttall :  Centralblatt  fur  Bakteriologie  nnd  Parasitenkunde,  1897, 
Abt.  i.,  Band  xxii.  p.  97. 


296  BACTERIOLOGY. 

hours  the  animal  is  quiet,  the  hair  is  rumpled,  tears 
stream  from  the  eyes,  and  later  convulsions  set  in,  which 
last  till  death.  The  results  found  at  autopsy  are  :  blood- 
stained oedema  at  the  site  of  inoculation,  reddening  and 
swelling  of  the  lymphatic  glands,  bloody  extravasation 
into  the  abdominal  walls,  serous  effusion  into  the  pleu- 
ral  and  peritoneal  cavities ;  the  intestine  is  occasionally 
hypersemic,  the  adrenal  bodies  congested,  and  the  spleen 
enlarged,  often  being  studded  with  grayish  points,  sug- 
gestive of  miliary  tubercles.  The  plague,  or  pest,  ba- 
cillus is  detected  in  large  numbers  in  the  local  oedema, 
the  lymph-glands,  the  blood,  and  the  internal  organs. 

As  is  the  case  with  the  group  of  hemorrhagic  septi- 
caemia bacteria,  the  members  of  which  it  resembles 
in  certain  other  respects,  when  death  does  not  result 
promptly  after  infection  there  is  usually  only  local  evi- 
dence of  the  inoculation,  the  distribution  of  the  micro- 
organisms throughout  the  body  being  considerably 
diminished. 

Animals  that  survive  inoculation  with  this  organism 
usually  exhibit  a  certain  degree  of  immunity  from  sub- 
sequent infection. 

Nuttall l  notes  that  feeding-experiments  have  resulted 
in  fatal  infection  in  gray  and  white  rats,  house-  and 
field-mice,  guinea-pigs,  rabbits,  hogs,  apes,  cats,  chick- 
ens, sparrows,  and  flies.  He  also  calls  attention  to  the 
fact  that  flies  may  live  for  several  days  after  being  in- 
fected with  this  organism,  and  if  at  liberty  to  fly  about 
may  manifestly  infect  persons  or  food-stuffs  on  which 
they  alight  or  fall. 

The  bacilli  apparently  lose  their  virulence  after 
long-continued  cultivation  under  artificial  conditions, 
1  Nuttall :  loc.  cit. 


THE  BACILLUS  OF  BUBONIC  PLAGUE.        297 

and  it  is  said  that  from  slowly  developing,  chronic 
buboes  non-virulent  or  feebly  virulent  cultures  are 
often  obtained.  Variations  in  the  degree  of  virulence 
have  been  observed  in  different  colonies  from  the  same 
source.  Virulence  is  said  by  Yersin,  Calmette,  and 
Borrel1  to  be  accentuated  by  passing  the  organism 
through  a  series  of  susceptible  animals. 

In  man  the  bacilli  are  most  numerous  in  the  en- 
larged, suppurating  lymphatics.  They  are  present,  but 
in  smaller  numbers,  in  the  blood  and  the  internal 
organs. 

It  has  been  observed  that  in  the  suppurating  lym- 
phatic glands  of  man  a  variety  of  organisms  may  be 
present,  but  among  them  are  always  the  plague  bacillus. 
Occasionally,  micrococci  predominate.  In  these  cases 
of  mixed  infection  the  pest  bacilli  are  said  to  stain  less 
intensely  with  alkaline  methylene-blue  than  do  the 
streptococci,  and  more  intensely  than  do  the  staphylo- 
cocci  that  are  present.  Also,  in  this  event,  the  strepto- 
cocci retain  the  Gram  stain,  while  the  pest  bacilli  do  not 
and  the  staphylococci  may  or  may  not.  It  has  been 
suggested  that  possibly  the  organisms  found  by  Kitasato 
in  the  blood,  and  which  he  describes  as  pest  bacilli, 
that  retained  the  color  when  treated  by  the  method  of 
Gram,  were  pairs  of  micrococci,  and  not  bacilli  at  all. 

It  is  the  opinion  of  Aoyama  that  the  suppuration  of 
the  glands  is  not  caused  by  the  plague  bacillus,  but  is 
rather  the  result  of  the  action  of  the  pyogenic  cocci 
with  which  it  is  so  often  associated.  It  is  also  his 
belief  that  the  most  important  and  frequent  mode  of 
infection  in  man  is  through  wounds  of  the  skin.  He 
does  not  regard  either  the  air-passages  or  the  alimentary 

1  Annales  de  1'Institut  Pasteur,  1895,  p.  589. 


298  BACTERIOLOGY. 

tract  as  frequent  portals  of  infection.  Wilm,  on  the 
contrary,  is  inclined  to  regard  the  alimentary  tract  as  a 
frequent  portal  of  infection.1 

The  order  in  which  the  lymphatics  manifest  disease 
appears  to  depend  upon  the  location  of  the  primary 
infection.  That  is  to  say,  if  it  is  upon  the  feet,  as  of 
persons  who  go  barefooted,  the  superficial  and  deep 
inguinal  glands  are  the  first  to  show  signs  of  the  dis- 
ease; while  if  infection  occurs  through  wounds  of  the 
hand,  the  buboes  appear  first  in  the  axillary  region. 
As  a  rule,  the  wound  through  which  infection  is  re- 
ceived shows  little  or  no  inflammatory  reaction.2 

Wyssokowitz  and  Zabolotny3  call  attention  to  the 
fact  that  the  blood  of  patients  convalescing  from  plague 
has  an .  agglutinating  action  upon  fluid  cultures  of  the 
plague  bacillus  analogous  to  that  observed  when  the 
blood-serum  of  typhoid  or  of  cholera  patients  is  mixed 
with  similar  cultures  of  the  typhoid  or  the  cholera 
bacillus. 

Yersin,  Calmette,  and  Borrel 4  have  demonstrated  that 
the  general  principles  underlying  the  establishment  of 
artificial  immunity  apply  as  well  to  this  disease  as  to 
a  number  of  others.  They  have  shown  that  by  the  use 
of  dead  cultures  (destroyed  by  heat)  of  the  plague 
bacillus  animals  may  be  rendered  immune  from  infec- 
tion by  the  virulent  living  organism.  They  have  also 
shown  that  the  serum  of  the  blood  of  these  animals  is 

1  Wilm :  loc.  cit. 

2  The  works  of  Yersin,  of  Kitasato,  and  of  Aoyama  have  been  ex- 
haustively reviewed  by  Flexner  in  the  Bulletin  of  the  Johns  Hopkins 
Hospital,  1894,  vol.  v.  p.  96,  and  1896,  vol.  vii.  p.  180.     I  am  indebted 
to  these  reviews  for  much  that  is  here  presented  on  this  subject. 

3Annales  de  1'Institut  Pasteur,  1897,  p.  663. 
4Ebenda,  1895,  p.  589. 


THE  BACILLUS  OF  BUBONIC  PLAGUE.        299 

not  only  capable  of  conferring  immunity  upon  other  ani- 
mals into  which  it  is  injected,  but  it  has  curative  proper- 
ties as  well,  providing  it  be  employed  at  an  early  stage  of 
the  disease.  In  1896  Yersin l  used  the  serum  of  arti- 
ficially immunized  horses  in  the  treatment  of  plague 
in  human  beings.  Of  26  persons  (3  in  Canton  and  23 
in  Amoy,  China)  who  received  injections  of  the  serum 
during  the  early  stages  of  the  disease,  in  no  case  later 
than  the  fifth  day,  only  2  died.  Comparing  this  mor- 
tality of  7.6  per  cent,  with  the  mortality  of  80  per  cent, 
among  persons  in  this  epidemic  who  were  treated  in 
other  ways,  he  feels  justified  in  regarding  the  method  as 
worthy  of  consideration. 

iEbenda,  1897,  p.  81. 


CHAPTER    XVII. 

Sputum  septicaemia — Septicaemia  resulting  from  the  presence  of  micro- 
coccus  tetragenus  in  the  tissues. 

OBTAIN  from  a  tuberculous  patient  a  sample  of  fresh 
sputum — that  of  the  morning  is  preferable.  Spread 
it  in  a  thin  layer  upon  a  black  glass  plate  and  select 
one  of  the  small,  white,  cheesy  masses  or  dense  mu- 
cous clumps  scattered  through  it.  With  a  pointed 
forceps  smear  this  carefully  upon  two  or  three  thin 
cover-slips,  dry  and  fix  them  in  the  way  given  for 
ordinary  cover-slip  preparations.  Stain  one  in  the 
ordinary  way  with  Loffler's  alkaline  methylene-blue 
solution,  one  other  by  the  Gram  method,  and  a  third 
after  the  method  given  for  tubercle  bacilli  in  fluids  or 
sputum. 

In  that  stained  by  Loffler's  method — slip  No.  1 — 
will  be  seen  a  great  variety  of  organisms — round  cells, 
ovals,  short  and  long  rods,  perhaps  spiral  forms.  But 
not  infrequently  will  be  seen  diplococci  having  more 
or  less  of  a  lancet  shape,  joined  together  by  their 
broad  ends,  the  points  of  the  lancet  being  away  from 
the  point  of  juncture  of  the  two  cells.  There  may 
also  be  seen  masses  of  cocci  which  are  conspicuous  by 
their  arrangement  into  groups  of  fours,  the  adjacent 
surfaces  being  somewhat  flattened.  They  are  not  sar- 
cina,  as  one  can  see  by  the  absence  of  the  division  in 
the  third  direction  of  space — they  divide  in  only  two 
directions. 

300 


SPUTUM  SEPTIC&MIA.  301 

In  the  slip  stained  by  the  Gram  method  the  same 
groups  of  cocci  which  grow  as  threes  and  fours 
will  be  seen;  but  the  lancet-shaped  diplococci  now 
present  an  altered  appearance — they  are  usually  sur- 
rounded by  a  capsule.  This  capsule  is  very  deli- 
cate in  structure,  and,  though  a  frequent  accompani- 
ment, is  not  constant.  It  can  sometimes  be  demon- 
strated by  the  ordinary  methods  of  staining,  though 
the  method  of  Gram  is  most  satisfactory.  (Fig.  63.) 

In  the  third  slip,  which  has  been  stained  by  the 
method  given  for  tubercle  bacilli  in  sputum,  if  decolor- 
ization  has  been  properly  conducted  and  no  contrast- 
stain  has  been  employed,  the  field  will  be  colorless  or 
of  only  a  very  pale  rose  color.  None  of  the  numerous 
organisms  seen  in  the  first  slip  can  now  be  detected ; 
but  instead  there  will  be  seen  scattered  through  the 
field  very  delicate  stained  rods,  which  present,  in 
most  instances,  a  conspicuous  beading  of  their  pro- 
toplasm— that  is,  the  staining  is  not  homogeneous, 
but  at  tolerably  regular  intervals  along  each  rod  are 
seen  alternating  stained  and  unstained  points.  These 
rods  may  be  found  singly,  in  groups  of  twos  and  threes, 
and  sometimes  in  clumps  consisting  of  large  numbers. 
\Yhen  in  twos  or  threes  it  is  not  uncommon  to  find 
them  describing  an  X  or  a  V  in  their  mode  of  arrange- 
ment, or  again  they  may  be  seen  lying  parallel  the  one 
to  the  other. 

If  contrast-stains  are  used,  these  rods  will  be  detected 
and  recognized  by  their  retaining  the  original  color 
with  which  they  have  been  stained ;  whereas  all  other 
bacteria  in  the  preparation,  as  well  as  the  tissue-cells 
which  are  in  the  sputum,  will  take  up  the  contrast- 
color.  (Fig.  62.) 


302  BACTERIOLOGY. 

This  delicate  beaded  rod  is  bacillus  tuberculosis.    The 
lancet-shaped  diplococcus  with  the  capsule  is  micrococcus 


FIG.  62. 


*  ^Itii  ' 


Tuberculous  sputum  stained  by  Gabbett's  method.    Tubercle  bacilli  seen  as 
red  rods ;  all  else  is  stained  blue. 

lanceolatus.  The  cocci  grouped  in  fours  are  micrococcus 
tetragenus. 

INOCULATION  EXPERIMENT. — Inoculate  into  the 
subcutaneous  tissues  of  a  guinea-pig  one  of  the  small, 
white,  caseous  masses  similar  to  that  which  has  been 
examined  microscopically.  If  death  ensues,  it  will,  in 
all  probability,  be  the  result  of  one  of  the  three  follow- 
ing forms  of  infection  : 

a.  Septicaemia  l  resulting  from  the  introduction  into 
the  tissues  of  an  organism  frequently  present  in  the 
sputum.  It  is  designated  variously  as  micrococcus  of 
sputum  septicaemia,  diplococcus  pneumonice,  pneumo- 
coccus  of  Frankel,  meningococcus,  streptococcus  lan- 
ceolatus Pasteuri,  micrococcus  lanceolatus,  miwococcus 
Pasteuri,  coccus  lanceolatus,  bacillus  salivarius  septicus, 

1  Septicaemia  is  that  form  of  infection  in  which  the  blood  is  the  chief 
field  of  activity  of  the  organisms. 


SPUTUM  SEPTICAEMIA.  303 

bacillus  septicus  sputigenus,  diplococcus  lanceolatus  cap- 
sulatusy  micrococcus  pneumonice  crouposce. 

b.  A   form   of  septicaemia   resulting   from   invasion 
of  the  tissues  by  an  organism  frequently  seen   in  the 
sputum   of  tuberculous   subjects.     It  is   characterized 

Sby  its  tendency  to  divide   into   fours.      It  is  micro- 
coccus  tetragenus. 

c.  Local  or  general  tuberculosis. 

a.   SPUTUM   SEPTIO^MIA. 

If  at  the  end  of  twenty-four  to  thirty-six  hours  the 
animal  be  found  dead,  we  may  reasonably  predict  that 
the  result  was  produced  by  the  introduction  into  the  tis- 
sues of  the  organism  of  sputum  septicaemia  above  men- 
tioned, viz.,  micrococcus  lanceolatuSj  which  is  not  uncom- 
monly found  in  the  mouths  of  healthy  individuals  as 
well  as  in  other  conditions. 

Inspection  of  the  seat  of  inoculation  usually  reveals 
a  local  reaction.  "  This  may  be  of  a  serous,  fibrinous, 
hemorrhagic,  necrotic,  or  purulent  character.  Fre- 
quently we  may  find  combinations  of  these  conditions, 
such  as  fibro-purulent,  fibri no-serous,  or  sero-hemor- 
rhagic."  *  The  most  conspicuous  naked-eye  change 
undergone  by  the  internal  organs  will  be  enlargement 
of  the  spleen.  It  is  usually  swollen,  but  may  at  times 
be  normal  in  appearance.  It  is  sometimes  hard,  dark 
red,  and  dry ;  or  it  may  be  soft  and  rich  in  blood.  Fre- 
quently there  is  a  limited  fibrinous  exudation  over  por- 
tions of  the  peritoneum. 

Except  in  the  exudations,  the  organisms  are  found 
only  in  the  lumen  of  the  bloodvessels,  where  they  are 

1  Welch  :  Johns  Hopkins  Hospital  Bulletin,  December,  1892,  vol.  iii. 
No.  27. 


304  BACTERIOLOGY. 

usually  present  in  enormous  numbers.  In  the  blood 
they  are  practically  always  free,  and  are  but  rarely  found 
within  the  bodies  of  leucocytes. 

In  stained  preparations  from  the  blood  and  exudates 
a  capsule  is  not  infrequently  seen  surrounding  the  organ- 
isms. (Fig.  63.)  This,  however,  is  not  constant. 


FIG.  63. 


Micrococcus  lanceolatus  in  blood  of  rabbit.    Stained  by  method  of  Gram. 
Decolorization  not  complete. 

If  a  drop  of  blood  from  the  dead  animal  be  intro- 
duced into  the  tissues  of  a  second  animal  (mouse  or 
rabbit),  identically  the  same  conditions  will  be  repro- 
duced. 

If  the  organism  be  isolated  in  pure  culture  from  the 
blood  of  the  animal,  and  a  portion  of  this  culture  be 
introduced  into  the  tissues  of  a  susceptible  animal, 
we  shall  see  again  the  same  pathological  picture. 

It  must  be  remembered,  however,  that  this  or- 
ganism when  cultivated  for  a  time  on  artificial  media 
rapidly  loses  its  pathogenic  properties.  If,  therefore, 
failure  to  reproduce  the  disease  after  inoculation 
with  old  cultures  should  occur,  it  is  in  all  probability 
due  to  a  loss  of  virulence  of  the  organism. 


SPUTUM  SEPTICAEMIA.  305 

This  organism  was  discovered  by  Sternberg  in  1880. 
It  was  subsequently  described  by  A.  FrankeL  as  the 
ctiological  factor  in  the  production  of  acute  fibrinous 
pneumonia. 

It  is  not  uncommonly  present  in  the  saliva  of  healthy 
individuals,  having  been  found  by  Sternberg  in  the  oral 
cavities  of  about  20  per  cent,  of  healthy  persons  examined 
by  him,  and  certain  authors  are  of  the  opinion  that  it 
occurs  in  the  oral  or  nasal  cavities  of  all  individuals 
at  various  times  during  life.  It  is  constantly  to  be 
detected  in  the  rusty  sputum  of  patients  suffering  from 
acute  fibrinous  pneumonia.  Its  presence  has  been  de- 
tected in  the  middle  ear,  in  the  pericardial  sac,  in  the 
pleura,  and  in  the  serous  cavities  of  the  brain ;  and 
indeed  it  may  penetrate  from  its  usual  site  of  develop- 
ment in  the  mouth  to  any  of  the  more  distant  organs. 

The  organism  is  commonly  found  as  a  diplococcus, 
though  here  and  there  short  chains  of  four  to  six  indi- 
viduals may  be  detected.  (Fig.  63.)  The  individual 
cells  are  more  or  Jess  oval,  or,  more  strictly  speaking, 
lancet-shaped,  for  at  one  end  they  are  commonly  pointed, 
When  joined  in  pairs  the  junction  is  always  at  the 
broad  ends  of  the  ovals,  never  at  the  pointed  extremities. 
When  in  chains  only  the  terminal  cells  are  pointed,  and 
then  at  their  distal  extremities. 

As  already  stated,  in  preparations  directly  from  the 
sputum  or  from  the  blood  of  animals  a  delicate  capsule 
may  frequently  be  seen  surrounding  them.  Though 
fairly  constant  in  preparations  directly  from  the  blood 
of  animals  and  from  the  sputum  or  lungs  of  pneumonic 
patients,  the  capsule  is  but  rarely  observed  in  artificial 
cultures.  Occasionally  in  cultures  on  blood-serum,  in 
milk,  and  on  agar-agar  it  can,  according  to  some 
20 


306  BACTERIOLOGY. 

authors,  be  detected ;  but  this  is  by  no  means  constant, 
or  even  frequent. 

Even  under  the  most  favorable  artificial  conditions 
this  organism  grows  but  slowly  and  frequently  not  at 
all. 

When  successfully  grown  upon  the  different  media  it 
presents  somewhat  the  following  appearances  : 

On  gelatin  its  development  is  very  limited  and  often 
no  growth  at  all  occurs.  This  is  probably  due  in  part 
to  the  low  temperature  at  which  gelatin  cultures  must 
be  kept.  If  development  occurs,  the  growth  appears 
as  minute  whitish  or  blue- white  points  on  the  plates. 
These  very  small  colonies  are  round,  finely  granular, 
sharply  circumscribed,  and  slightly  elevated  above  the 
surface  of  the  gelatin.  They  do  not  cause  liquefaction 
of  the  gelatin. 

If  grown  in  slant-  or  stab-cultures,  the  surface-devel- 
opment is  very  limited ;  along  the  needle-track  tiny 
whitish  or  bluish-white  granules  appear. 

On  nutrient  agar-agar  the  colonies  are  almost  trans- 
parent, more  or  less  glistening,  and  very  delicate  in 
structure.  On  blood-serum  development  is  more 
marked,  though  still  extremely  feeble,  appearing  as 
a  cluster  of  isolated  fine  points  growing  closely  side  by 
side. 

Growth  on  potato  is  not  usually  observed. 

When  grown  in  milk  it  commonly  causes  an  acid 
reaction  with  coincident  coagulation  of  the  casein. 
Some  varieties,  especially  non-virulent  ones,  do  not 
coagulate  milk.1 

It  is  not  motile. 

It  grows  best  at  a  temperature  of  from  35°  to  38°  C. 

1  Welch :  loc.  cit. 


SPUTUM  SEPTICAEMIA.  307 

Below  24°  C.  there  is  usually  no  development,  but  in  a 
few  cases  it  has  been  seen  to  grow  at  as  low  a  tempera- 
ture as  18°  C.  Above  42°  C.  development  is  checked. 

It  grows  as  well  without  as  with  oxygen.  It  is 
therefore  one  of  the  facultative  anaerobic  forms. 

Cultivation  of  this  organism  is  most  successful  when 
the  agar-agar-gelatin  mixture  of  Guarniari  is  employed. 
(See  this  medium.) 

It  may  be  stained  with  the  ordinary  aniline  staining- 
reagents.  For  demonstrating  the  capsule  the  method 
of  Gram  and  the  acetic-acid  method  give  the  best  re- 
sults. (See  Stainings.) 

This  organism  is  conspicuous  for  the  irregularity  of 
its  behavior  when  grown  under  artificial  conditions : 
usually  it  loses  its  pathogenic  properties  after  a  few 
generations ;  but  again  this  peculiarity  may  be  retained 
for  a  much  longe'r  time.  Not  rarely  it  fails  to  grow 
after  three  or  four  transplantations  on  artificial  media, 
though  at  times  it  may  be  carried  through  many  gen- 
erations. 

INOCULATION  INTO  ANIMALS. — The  results  of  inocu- 
lations with  pure  cultures  of  this  organism  are  also  con- 
spicuous for  their  irregularity.  When  the  organism  is 
of  full  virulence  the  form  of  septicaemia  just  described 
is  usually  produced,  but  at  times  it  is  found  to  be  totally 
devoid  of  pathogenic  powers :  between  these  extremes 
cultures  may  be  obtained  possessing  every  variation  in 
the  intensity  of  their  disease-producing  properties. 
The  principal  pathological  conditions  that  may  be  pro- 
duced by  the  inoculation  of  susceptible  animals  with 
this  organism  are,  according  to  the  degree  of  its  viru- 
lence, acute  septicaemia,  spreading  inflammatory  exuda- 
tions, and  circumscribed  abscesses.  All  three  of  these 


308  BACTERIOLOGY. 

conditions  may  sometimes  be  produced  by  inoculating 
rabbits  with  the  same  cultures  in  varying  amounts. 

Rabbits,  mice,  guinea-pigs,  dogs,  rats,  cats,  and  sheep 
are  susceptible  to  infection  by  this  organism.  Chickens 
and  pigeons  are  insusceptible.  Young  animals,  as  a 
rule,  are  more  easily  infected  than  old  ones.  Rabbits 
and  mice  are  the  most  susceptible  of  the  animals  used 
for  experimental  purposes,  and  in  testing  the  virulence 
of  a  culture  it  is  well  to  inoculate  one  of  each,  for  the 
same  culture  may  sometimes  be  virulent  for  mice  and 
not  for  rabbits,  and  viee  versa. 

If  the  culture  is  virulent,  in tra vascular  or  intra- 
peritoneal  injections  into  rabbits  may  produce  rapid  and 
fatal  septicaemia ;  while  subcutaneous  inoculation  of  the 
same  material  may  result  in  only  a  localized  inflamma- 
tory process.  On  the  other  hand,  subcutaneous  inocula- 
tion of  less  virulent  cultures  may  produce  a  local  process, 
while  intravenous  inoculation  may  be  without  result. 
This  organism  is  the  cause  of  a  number  of  pathological 
conditions  in  human  beings  that  have  not  hitherto  been 
considered  as  related  to  one  another  etiologically.  It 
is  always  present  in  the  inflamed  area  of  the  lung  in 
acute  fibrinous  or  lobar  pneumonia  ;  it  is  known  to  cause 
acute  cerebro-spinal  meningitis,  endo-  and  peri  -carditis, 
certain  forms  of  pleuritis,  arthritis  and  peri-arthritis, 
and  otitis  media. 

6.   SEPTICAEMIA  CAUSED  BY  MICROCOCCUS  TETRAGENTJS. 

Should  the  death  of  the  animal  not  occur  within  the 
first  twenty-eight  to  thirty  hours  after  inoculation,  but 
be  postponed  until  between  the  fourth  and  eighth  day,  it 
may  result  from  the  invasion  of  the  tissues  by  the  organ- 
ism now  to  be  described,  viz.,  microeoccus  tetragenus. 


. 

MICROCOCCUS  TETRAGENUS.  309 

This  organism  was  discovered  by  Gaffky,  and  was 
subsequently  described  by  Koch*  in  the  account  of  his 
experiments  upon  tuberculosis.  It  is  often  present  in 
the  saliva  of  healthy  individuals  and  is  commonly 
present  in  the  sputum  of  tuberculous  patients.  Koch 
found  it  very  frequently  in  the  pulmonary  cavities  of 
phthisical  patients.  It,  however,  plays  no  part  in  the 
etiology  of  tuberculosis. 

It  is  a  small  round  coccus  of  about.  1  //  transverse 
diameter.  It  is  seen  as  single  cells,  joined  in  pairs, 
and  in  threes ;  but  its  most  conspicuous  grouping  is  in 
fours,  from  which  arrangement  it  takes  its  name.  In 
preparations  made  from  cultures  of  this  organism  it 
is  not  rare  to  find,  here  and  there,  single  bodies  which 
are  much  larger  than  the  other  individuals  in  the  field. 
Close  inspection  reveals  them  to  be  cells  in  the  initial 
stage  of  division  into  twos  and  fours.  A  peculiarity 
of  this  organism  is  that  the  cells  are  bound  together 
by  a  transparent  gelatinous  mass. 

When  cultivated  artificially  it  grows  very  slowly. 

Upon  gelatin  plates  the  colonies  appear  as  round, 
sharply  circumscribed,  punctiform  masses  which  are 
slightly  elevated  above  the  surface  of  the  surrounding 
medium.  Under  a  low  magnifying  power  they  are  seen 
to  be  slightly  granular  and  to  present  a  more  or  less 
glassy  lustre. 

The  colonies  increase  but  little  in  size  after  the  third 
or  fourth  day.  If  cultivated  as  stab-cultures  in  gelatin, 
there  appears  upon  the  surface  at  the  point  of  inocula- 
tion a  circumscribed  white  point,  slightly  elevated  above 
the  surface  and  limited  to  the  immediate  neighborhood 
of  the  point  of  inoculation.  Down  the  needle-track  the 
growth  is  not  continuous,  but  appears  in  isolated,  round, 


310  BACTERIOLOGY. 

dense  white  clumps  or  beads,  which  do  not  develop 
beyond  very  small  points. 

It  does  not  liquefy  gelatin. 

Upon  plates  of  nutrient  agar-agar  the  colonies  appear 
as  small,  almost  transparent,  round  points,  which  have 
about  the  same  color  and  appearance  as  a  drop  of  egg- 
albumin;  they  are  very  slightly  opaque.  They  are 
moist  and  glistening.  They  rarely  develop  to  an  extent 
exceeding  1  to  2  mm.  in  diameter. 

Upon  agar-agar  as  stab-  or  slant-cultures  the  surface- 
growth  has  more  or  less  of  a  mucoid  appearance.  It 
is  moist,  glistening,  and  irregularly  outlined.  The  out- 
line of  the  growth  depends  upon  the  moisture  of  the 
agar-agar.  It  is  slightly  elevated  above  the  surface  of 
the  medium. 

In  contradistinction  to  the  gelatin  stab-cultures,  the 
growth  in  agar-agar  is  continuous  along  the  track  of 
the  needle. 

The  growth  on  potato  is  a  thick,  irregular,  slimy- 
looking  patch. 

The  transparent  mucilaginous  substance  which  is  seen 
to  surround  these  organisms  renders  them  coherent,  so 
that  efforts  to  take  up  a  portion  of  a  colony  from  the 
agar-agar  or  potato  cultures  result  usually  in  drawing 
out  fine,  silky  threads,  consisting  of  organisms  imbedded 
in  the  mucoid  material. 

The  organism  grows  best  at  from  35°  to  38°  C.,  but 
can  be  cultivated  at  the  ordinary  room-temperature — 
about  20°  C. 

The  growth  under  all  conditions  is  slow. 

It  grows  both  in  the  presence  of  and  without  oxygen. 

It  is  not  motile. 

It  stains  readily  with  all  the  ordinary  aniline  dyes. 


MICROCOCCUS  TETRAGENUS.  311 

In  tissues  its  presence  is  readily  demonstrated  by  the 
stain  ing-method  of  Gram. 

The  grouping  into  fours  is  particularly  well  seen  in 
sections  from  the  organs  of  animals  dead  of  this  form 
of  septicaemia.  In  such  sections  the  organisms  will 
always  be  found  within  the  capillaries. 

INOCULATION  INTO  ANIMALS.— To  the  naked  eye 
no  alteration  can  be  seen  in  the  organs  of  animals  that 
have  died  as  a  result  of  inoculation  with  micrococcm 
tdragenus;  but  microscopic  examination  of  cover-slip 
preparations  from  the  blood  and  viscera  reveals  the 
presence  of  the  organisms  throughout  the  body — espe- 
cially is  this  true  of  preparations  from  the  spleen. 
White  mice  and  guinea-pigs  are  susceptible  to  the  dis- 
ease. Gray  mice,  dogs,  and  rabbits  are  not  susceptible 
to  this  form  of  septicaemia.  Subsequent  inoculation  of 
healthy  animals  with  a  drop  of  blood,  a  bit  of  tissue,  or 
a  portion  of  a  pure  culture  of  this  organism  from  the 
body  of  an  animal  dead  of  this  disease,  results  in  a  re- 
production of  the  conditions  found  in  the  dead  animal 
from  which  the  tissues  or  cultures  were  obtained. 

It  sometimes  happens  that  in  guinea-pigs  which  have 
been  inoculated  with  this  organism  local  pus-formations 
result,  instead  of  a  general  septicaemia.  The  organisms 
will  then  be  found  in  the  pus-cavity. 


CHAPTER  XVIII. 

Tuberculosis — Microscopic  appearance  of  miliary  tubercles — Encap- 
sulation of  tuberculous  foci — Diffuse  caseation — Cavity-formation 
— Primary  infection — Modes  of  infection — Location  of  the  bacilli 
in  the  tissues — Staining-peculiarities — Organisms  with  which 
bacillus  tuberculosis  may  be  confounded — Points  of  differentiation — 
Bacillus  of  influenza. 

C.  LOCAL  OR  GENERAL  TUBERCULOSIS. 

SHOULD  the  animal  succumb  to  neither  of  the  septic 
processes  just  described,  then  its  death  from  tuberculosis 
may  be  reasonably  expected. 

When  this  disease  is  in  progress  alterations  in  the 
lymphatic  glands  nearest  the  site  of  inoculation  may 
be  detected  by  the  touch  in  from  two  to  four  weeks. 
They  will  then  be  found  enlarged.  Though  not  con- 
stant, tumefaction  and  subsequent  ulceration  at  the 
point  of  inoculation  may  be  observed.  Progressive 
emaciation,  loss  of  appetite,  and  difficulty  in  respiration 
point  to  the  existence  of  the  general  tuberculous  process. 
Death  ensues  in  from  four  to  eight  weeks  after  inocula- 
tion. At  autopsy  either  general  or  local  tuberculosis 
may  be  found.  The  expressions  of  tuberculosis  are  so 
manifold  and  in  different  animals  vary  so  widely  the 
one  from  the  other,  that  no  fixed  law  as  to  what  will 
appear  at  autopsy  can  d,  priori  be  laid  down. 

The  guinea-pig,  which  is  best  suited  for  this  experi- 
ment because  its  susceptibility  to  tuberculosis  is  greater 
and  more  constant  than  that  of  other  animals  usually 
312 


LOCAL   OR  GENERAL  TUBERCULOSIS.         313 

found  in  the  laboratory,  presents,  in  the  main,  changes 
that  are  characterized  by  coagulation-necrosis  'and  case- 
ation.  This  is  particularly  the  case  when  the  infec- 
tion is  general — i.  e.,  when  the  process  is  of  the  acute 
miliary  type ;  then  the  tissues  of  the  liver  and  spleen 
present  the  most  favorable  field  for  the  study  of  this 
pathological-anatomical  alteration. 

In  general,  the  tubercular  lesions  can  be  divided  into 
those  of  strictly  focal  character — i.  e.,  the  miliary  and 
the  conglomerate  tubercles — and  those  which  are  more 
diffuse.  The  latter  lesions,  although  fundamentally 
of  the  same  nature  as  the  miliary  tubercles,  are  much 
greater  in  extent  and  not  so  sharply  circumscribed. 
These  latter  lesions  play  a  more  conspicuous  rdle  in  the 
pathology  of  the  disease  than  do  the  miliary  nodules, 
although  it  is  to  the  presence  of  the  miliary  nodules  that 
the  disease  owes  its  name. 

At  autopsy  the  pathological  manifestations  of  the  dis- 
ease are  not  infrequently  seen  to  be  confined  to  the  seat 
of  inoculation  and  to  the  neighboring  lymphatic  glands. 
These  tissues  then  present  all  the  characteristics  of  the 
tuberculous  process  in  the  stage  of  cheesy  degeneration. 
When  the  disease  is  more  general  the  degree  of  its  exten- 
sion varies.  Sometimes  the  small  gray  nodules — mili- 
ary tubercles — are  only  to  be  seen  with  the  naked  eye  in 
the  tissues  of  the  liver  and  spleen.  Again,  they  may  in- 
vade the  lung,  and  frequently  they  are  distributed  over 
the  serous  membranes  of  the  intestines,  the  lungs,  the 
heart,  and  the  brain.  These  gray  nodules,  as  seen  by 
the  naked  eye,  vary  in  size  from  that  of  a  pin-point  to 
that  of  a  hempseed,  and,  as  a  rule,  are,  in  this  stage,  the 
result  of  the  fusion  of  two  or  more  smaller  miliary  foci. 
Though  the  two  terms  "  miliary  "  and  "  conglomerate  " 


314  BACTERIOLOGY. 

are  employed  for  the  description  of  the  microscopic 
appearance  of  these  nodules,  yet  it  is  very  rarely  that 
any  condition  other  than  that  due  to  the  fusion  of 
several  of  these  minute  foci  can  be  detected  by  the 
naked  eye. 

The  miliary  tubercles  are  of  a  pale  gray  color,  with  a 
white  centre,  are  slightly  elevated  above  the  surface  of 
the  tissue  in  which  they  are  located,  and,  as  stated,  vary 
considerably  in  dimensions,  usually  appearing  as  points 
which  range  in  size  from  that  of  a  pin-point  to  that  of 
a  pin-head.  They  are  not  only  located  upon  the  surface 
of  the  organs,  but  are  distributed  through  the  depths  of 
the  tissues.  To  the  touch  they  sometimes  present  noth- 
ing characteristic,  but  when  closely  packed  together  in 
large  numbers  they  usually  give  a  mealy  or  sandy  sen- 
sation to  the  hand  passed  over  them.  Stained  sections 
of  miliary  tubercles  present  a  distinctly  characteristic 
appearance,  and  the  disease  may  be  diagnosticated  by 
these  histological  changes  alone,  though  the  crucial  test 
in  the  diagnosis  is  the  demonstration  of  tubercle  bacilli 
in  these  nodules. 

MICROSCOPIC  APPEARANCE  OF  MILIARY  TUBER- 
CLES.— A  miliary  tubercle  under  a  low  magnifying 
power  of  the  microscope  presents  somewhat  the  follow- 
ing appearance :  there  is  a  central  pale  area,  evidently 
composed  of  necrotic  tissue  because  of  its  incapacity 
for  taking  up  the  nuclear  stains  commonly  employed. 
Scattered  through  this  necrotic  area  may  be  seen  granular 
masses  irregular  in  size  and  shape  ;  they  take  up  the  stains 
employed  and  are  evidently  fragments  of  cell-nuclei  in 
course  of  destruction.  Through  the  necrotic  area  may 
here  and  there  be  seen  irregular  lines,  bands,  or  ridges,  the 
remains  of  tissues  not  yet  completely  destroyed  by  the 


LOCAL  OR  GENERAL   TUBERCULOSIS.         315 

necrotic  process.  Around  the  periphery  of  this  area 
may  sometimes  be  noticed  large  multinuclealed  cells, 
the  nuclei  of  which  are  arranged  about  the  periphery 
of  the  cell  or  grouped  irregularly  at  its  poles.  The 
arrangement  of  these  nuclei  as  observed  in  sections 
is  usually  oval,  or  somewhat  crescentic.  In  tuber- 
cles from  the  human  subject  these  large  "  giant-cells," 
as  they  are  called,  are  quite  common.  They  are  much 
less  frequent  in  tubercular  tissues  from  lower  ani- 
mals. 

Round  about  the  central  focus  of  necrosis  is  seen  a 
more  or  less  broad  zone  of  closely  packed  small  round 
and  oval  bodies,  Avhich  stain  readily  but  not  homoge- 
neously. They  vary  in  size  and  shape,  and  are  seen  to 
be  imbedded  in  a  delicate  network  of  fibrinous-looking 
tissue.  This  fibrin-like  network  in  which  these  bodies>lie, 
and  which  is  a  common  accompaniment  of  giant-cell  for- 
mation, is  in  part  composed  of  fibrin,  but  is  in  the  main, 
most  probably,  the  remains  of  the  interstitial  fibrous 
tissue  of  the  part.  This  zone  of  which  we  are  speak- 
ing is  the  zone  of  so-called  "granulation-tissue,"  and 
consists  of  leucocytes,  granulation-cells,  fibrin,  and  the 
fibrous  remains  of  the  organ ;  the  irregularly  oval,  gran- 
ular bodies  which  take  up  the  stain  are  the  nuclei  of 
these  cells.  The  zone  of  granulation-tissue  surrounds 
the  whole  of  the  tuberculous  process,  and  at  its  periphery 
fades  gradually  into  the  healthy  surrounding  tissues  or 
fuses  with  a  similar  zone  surrounding  another  tubercu- 
lar focus.  This  may  be  taken  as  a  description  of  the 
typical  miliary  tubercle. 

DIFFUSE  CASEATION. — The  diffuse  caseation,  as  said, 
plays  a  more  important  role  in  the  tuberculous  lesion, 
both  in  the  human  and  experimental  forms,  than  does 


316  BACTERIOLOGY. 

the  formation  of  miliary  tubercles.  In  this  a  large 
area  of  tissue  undergoes  the  same  process  of  necrosis 
and  caseation  as  the  centre  of  the  miliary  tubercle. 
In  certain  tissues,  notably  the  lungs  and  lymphatics, 
it  is  more  marked  than  in  others.  In  rabbits,  par- 
ticularly, all  the  changes  in  the  4ung  frequently  come 
under  this  head.  When  this  is  the  case  solid  masses 
are  found,  sometimes  as  large  as  a  pea,  or  involving 
even  an  entire  lobe  or  the  whole  lung  in  some  cases. 
They  are  of  a  whitish-yellow,  opaque  color,  and  on  sec- 
tion are  peculiarly  dry  and  hard.  Entire  lymphatic 
glands  may  be  changed  in  this  way.  The  conditions 
which  appear  to  be  most  favorable  to  the  occurrence 
of  this  widespread  caseation  of  the  tissues  are  the 
simultaneous  deposition  of  a  large  number  of  tubercle 
bacilli  in  them,  and  the  involvement  of  a  wide  area  in- 
stead of  a  single  isolated  point,  as  in  the  miliary  tubercle. 
Necrosis  is  so  rapid  that  time  does  not  suffice  for  those 
reactive  changes  to  take  place  in  the  tissues  which  result 
in  the  formation  of  the  outer  zone  of  the  miliary  tubercle. 
In  other  instances  the  entire  caseous  area  is  surrounded 
by  a  granulation-zone  similar  to  that  around  the  caseous 
centre  of  the  miliary  tubercles.  It  is  of  special  im- 
portance to  recognize  the  etiological  connection  between 
this  diffuse  caseation  and  the  tubercle  bacillus,  because 
until  its  nature  was  accurately  determined  caseous  pneu- 
monia of  the  lungs  formed  the  chief  obstacle  which 
many  encountered  in  recognizing  the  specific  infectious- 
ness  of  tuberculosis. 

CAVITY-FOKMATION. — The  production  of  cavities,  a 
prominent  feature  in  human  tuberculosis,  particularly 
of  the  lungs,  is  due  to  softening  of  the  necrotic, 
caseous  masses  or  of  aggregations  of  miliary  tuber- 


LOCAL  OR  GENERAL  TUBERCULOSIS.         317 

cles.  The  material  softens  and  is  expelled,  and  a 
cavity  remains.  In  the  wall  of  this  cavity  the  tuber- 
culous changes  still  proceed,  both  as  diffuse  caseation 
and  formation  of  miliary  tubercles.  The  whole  cavity 
with  the  reactive  changes  in  the  tissues  of  its  walls 
may  be  properly  conceived  as  a  single  gigantic  tuber- 
cle, its  wall  forming  a  tissue  very  analogous  to  the  outer 
zone  of  the  single  tubercle,  the  cavity  itself  correspond- 
ing to  the  caseous  centre. 

In  animals  used  for  experiment  cavity-formation  of 
this  sort  is  very  rare,  owing  to  the  greater  resistance  of 
the  caseous  tissue.  That  it  is,  however,  possible  to  pro- 
duce in  rabbits  pulmonary  cavities  in  all  physical  re- 
spects similar  to  those  seen  in  the  human  being  has 
been  most  beautifully  demonstrated  by  Prudden.  He 
showed  that  when  he  had  injected  fluid  cultures  of  strep- 
tococcus pyogenes  into  the  trachea  of  rabbits  already  af- 
fected with  tubercular  consolidation  of  the  lungs,  the 
result  of  the  mixed  infection  thus  brought  about  was 
cavity-formation  in  eight  out  of  nine  lungs  subjected  to 
the  conditions  of  the  experiment ;  while  in  only  one  out 
of  eleven  did  cavities  form  under  the  influence  of  the 
tubercle  bacillus  alone.1 

In  the  contents  and  in  the  walls  of  tubercular  cavi- 
ties in  man  bacteria  other  than  bacillus  tuberculosis  are 
found.  It  is  to  the  influence  of  some  of  these,  as  we 
have  seen,  that  diseases  other  than  tuberculosis  may 
sometimes  be  produced  by  the  inoculation  of  animals 
with  the  sputum  from  such  cases. 

ENCAPSULATION   OF   TUBERCULAR   Foci. — It    not 


1  Prudden :  "  Experimental  Phthisis  in  Rabbits,  with  the  Formation 
of  Cavities,"  etc.,  Transactions  of  the  Association  of  American  Physi- 
cians, 1894,  vol.  ix.  p.  166. 


318  BACTERIOLOGY. 

uncommonly  occurs  that  round  about  a  necrotic  tuber- 
culous focus  there  is  formed  a  fibrous  capsule  which  may 
completely  shut  off  the  diseased  from  the  healthy  tissue 
surrounding  it ;  or  a  tuberculous  focus  may,  through  the 
resistance  of  the  tissue  in  which  it  is  located,  be  more 
or  less  completely  isolated.  In  this  condition  the  dis- 
eased foci  may  lie  dormant  for  a  long  time  and  give  no 
evidence  of  their  existence,  until  they  are  caused  to 
break  through  their  envelopes  by  some  disturbing 
cause.  With  the  passage  of  the  bacilli  or  their  spores 
from  such  a  focus  into  the  vascular  or  lymphatic  cir- 
culation the  disease  may  become  general. 

It  is  to  some  such  accident  as  this  that  the  sudden 
appearance  of  general  tubercular  infection  in  subjects 
supposed  to  have  recovered  from  the  primary  local 
manifestations  may  often  be  attributed.  The  breaking- 
down  of  old  caseous  lymphatic  glands  is  a  common 
example  of  this  recurrence  of  tuberculosis. 

PRIMARY  INFECTION.  —  Primary  infection  occurs 
through  either  the  vascular  or  lymphatic  circulation. 
Through  these  channels  the  bacilli  gain  access  to  the 
tissues  and  become  lodged  in  the  finer  capillary  ramifi- 
cations or  in  the  more  minute  lymph-spaces.  Here 
they  find  conditions  favorable  to  their  development, 
and  in  the  course  of  their  life-processes  produce  sub- 
stances of  a  chemical  nature  which  serve  to  bring 
about  the  death  of  the  tissues  in  their  immediate 
neighborhood.  This  tissue-death  is  probably  the  very 
first  effect  of  the  bacilli  in  the  body,  and  repre- 
sents the  necrotic  centre  which  can  always  be  seen  in 
even  the  most  minute  tubercles.  With  the  production 
of  this  progressive  necrosis — for  progressive  it  is,  as  it 
proceeds  as  long  as  the  bacilli  live  and  continue  to 


LOCAL  OR  GENERAL  TUBERCULOSIS.         319 

produce  their  poisonous  products — there  is  in  addition 
a  reactive  change  in  the  surrounding  tissue's,  which 
consists  in  the  formation  of  a  granulation-zone  at  the 
outer  margins  of  the  dying  and  dead  tissue.  This  zone 
consists  of  small,  round  granulation-cells  and  of  leuco- 
cytes, all  of  which  are  seen  in  the  meshes  of  the  finer 
fibrous  tissues  of  the  part.  At  the  same  time  altera- 
tions are  produced  in  the  walls  of  the  vessels  of  the 
locality  ;  these  tend  to  occlude  them,  and  thus  the  proc- 
ess of  tissue-death  is  favored  by  a  diminution  of  the 
amount  of  nutrition  brought  to  them.  These  changes 
may  continue  until  eventually  conglomerate  tubercles, 
widespread  caseation,  or  cavity-formation  results;  or 
from  one  cause  or  another  the  life-processes  of  the 
bacilli  may  be  checked  and  recovery  occur. 

MODES  OF  INFECTION. — Experimentally,  tuberculosis 
may  be  produced  in  susceptible  animals  by  subcutaneous 
inoculation,  by  direct  injection  into  the  circulation, 
by  injection  into  the  peritoneal  cavity,  by  feeding  of 
tuberculous  material,  by  the  introduction  of  the  bacilli 
into  the  air-passages,  and  by  inoculation  into  the  ante- 
rior chamber  of  the  eye. 

In  the  human  subject  the  most  common  portals  of 
infection  are,  doubtless,  the  air-passages,  the  alimentary 
tract,  and  cutaneous  wounds.  When  introckiced  subcu- 
taneously  the  resulting  process  finds  its  most  pronounced 
expression  in  the  lymphatic  system.  The  growing 
bacilli  make  their  way  into  the  lymphatic  spaces  of 
the  loose  cellular  tissue,  are  taken  up  in  the  lymph- 
stream  and  deposited  in  the  neighboring  lymphatic 
glands.  Here  they  may  remain  and  give  rise  to  no 
alteration  other  than  that  seen  in  the  glands  them- 
selves; or  they  may  pass  on  to  neighboring  glands, 


320  BA  CTERIOL  OGY. 

and  eventually  be  disseminated  throughout  the  lym- 
phatic system,  ultimately  reaching  the  vascular  system. 

Having  gained  access  to  the  bloodvessels  the  results 
are  the  same  as  those  following  intravascular  injection 
of  the  bacilli,  namely :  general  tuberculosis  quickly 
follows,  with  the  production  of  miliary  tubercles  most 
conspicuous  in  the  lungs  and  kidneys ;  less  numerous 
in  the  spleen,  liver,  and  bone-marrow. 

When  inhaled  into  the  lungs,  if  conditions  are  favor- 
able, multiplication  of  the  bacilli  quickly  follows.  Co- 
incident with  their  growth  they  are  mechanically  pressed 
into  the  tissues  of  the  lungs.  As  multiplication  con- 
tinues some  are  transported  from  the  primary  site  of 
infection  to  healthy  portions  of  the  lung-tissue,  where, 
through  their  development,  the  process  is  repeated. 

In  the  same  way  infection  by  way  of  the  alimentary 
tract  is  in  the  main  due  to  the  bacilli  being  forced  by 
mechanical  pressure  into  the  walls  of  the  intestines.  In- 
vestigation has  shown  that  lesions  of  the  intestinal  coats 
are  not  necessary  for  the  entrance  of  tubercle  bacilli 
from  the  lumen  of  the  intestines  into  the  internal 
organs  and  tissues.  They  may  be  transported  from 
the  intestinal  tract  into  the  lymphatics  in  the  same 
way  that  the  fat-droplets  of  the  chyle  find  entrance 
into  the  lymphatic  circulation. 

Unlike  most  pathogenic  organisms,  the  tubercle  ba- 
cillus is  believed  to  have  the  property  of  forming  spores 
within  the  tissues.  These  spores,  which  are  presum- 
ably highly  resistant  and  not  destroyed  by  drying, 
are  thrown  off  from  the  lungs  in  the  sputum  of  tuber- 
culous patients  in  large  numbers ;  and  unless  special 
precautions  be  taken  to  prevent  it,  the  sputum  becomes 
dried,  is  ground  into  dust,  and  sets  free  in  the  atmos- 


LOCAL   OR   GENERAL   TUBERCULOSIS.         321 

pherc  the  spores  of  tubercle  bacilli  which  came  with  it 
from  the  limgs,  and  which  have  the  property  of  ex- 
citing the  disease  in  a  certain  number  of  persons  who 
inhale  them.  The  frequency  of  pulmonary  tuber- 
culosis points  to  this  as  one  of  the  commonest  sources 
and  modes  of  infection  in  human  beings.  This  opinion 
is  borne  out  by  statistical  studies  upon  the  disease,  as 
well  as  by  such  evidence  as  Cornet  *  has  produced  upon 
the  infective  nature  of  dust  taken  from  apartments  occu- 
pied by  persons  suffering  from  pulmonary  tuberculosis. 

LOCATION  OF  THE  BACILLI  IN  THE  TISSUES. — The 
bacilli  will  be  found  most  numerous  in  those  tissues 
which  are  the  seats  of  the  active  stage  of  the  process. 

In  the  initial  stage  of  the  disease  the  bacilli  will 
be  fewer  in  number  than  later.  At  this  time  only 
single  rods  may  here  and  there  be  found  ;  later  they 
are  more  numerous ;  and,  finally,  when  the  process 
has  advanced  to  a  stage  easily  recognizable  by  the  naked 
eye,  they  are  found  in  the  granulation-zones  in  clumps 
and  scattered  about  in  large  numbers. 

In  the  central  necrotic  masses,  which  consist  of  cell- 
detritus,  it  is  rare  that  the  organisms  can  be  demon- 
strated microscopically.  It  is  at  the  periphery  of  these 
areas  and  in  the  progressing  granular  zone  that  they 
are  most  frequently  to  be  seen. 

This  apparent  absence  of  the  bacilli  from  the  central 
necrotic  area  and  often  from  old  caseous  tissues  must 
not  be  taken,  however,  as  evidence  that  these  materials 
are  not  infective.  As  bacilli,  they  are  difficult  to 
demonstrate  here  because  the  probabilities  are  that  at 
this  stage  of  the  process,  owing  to  conditions  unfavor- 
able to  their  further  growth,  they  are  in  the  spore- 

1  Cornet :  Zeitschrift  fur  Hygiene,  1889,  Bd.  v.  S.  191. 
21 


322  BACTERIOLOGY. 

stage,  a  stage  in  which  it  is  as  yet  impossible,  with 
our  present  methods  of  staining,  to  render  them  visi- 
ble. The  facts  that  this  tissue  is  infective,  and  that 
with  it  the  disease  can  be  reproduced  in  susceptible 
animals,  speak  for  the  accuracy  of  this  assumption. 
A  conspicuous  example  of  this  condition  is  seen  in 
old  scrofulous  glands.  These  glands  usually  present 
a  slow  process,  are  commonly  caseous,  and  always 
possess  the  property  of  producing  the  disease  when 
introduced  into  the  tissues  of  susceptible  animals,  and 
yet  they  are  the  most  difficult  of  all  tissues  in  which  to 
demonstrate  microscopically  the  presence  of  tubercle 
bacilli. 

In  tubercles  containing  giant-cells  the  bacilli  can 
usually  be  demonstrated  in  the  granular  contents  of 
these  cells.  Frequently  they  will  be  found  accumu- 
lated at  the  pole  of  the  cell  opposite  to  that  occupied 
by  the  nuclei,  as  if  there  existed  an  antagonism  between 
the  nuclei  and  the  bacilli.  In  some  of  these  cells, 
however,  the  distribution  of  the  bacilli  is  seen  to  be 
irregular,  and  they  will  be  found  scattered  among  the 
nuclei  as  well  as  in  the  necrotic  centre  of  the  cell.  As 
the  number  of  bacilli  in  the  giant-cell  increases  the  cell 
itself  is  ultimately  destroyed. 

Tubercular  tissues  always  contain  the  bacilli  or  their 
spores,1  and  are  always  capable  of  reproducing  the  dis- 
ease when  introduced  into  the  body  of  a  susceptible 
animal.  From  the  tissues  of  this  animal  the  bacilli 
may  be  obtained  and  cultivated  artificially,  and  these 
cultures  are  capable  of  again  producing  the  disease 
when  further  inoculated.  Thus  the  postulates  for- 
mulated by  Koch,  which  are  necessary  to  prove  the 
1  The  property  of  spore-formation  is  assumed,  not  proved. 


THE  TUBERCLE  BACILLUS.  323 

etiological  role  of  an  organism  in  the  production  of  a 
malady,  are  fulfilled. 

THE   TUBERCLE   BACILLUS. 

Of  the  three  pathogenic  organisms  liable  to  occur 
in  the  sputum  of  a  tuberculous  subject,  the  tubercle 
bacillus  gives  us  the  greatest  difficulty  in  our  efforts  at 
cultivation. 

It  is,  in  the  strict  sense  of  the  word,  a  parasite,  and 
finds  conditions  entirely  favorable  to  its  development 
only  in  the  animal  body.  On  ordinary  artificial  media 
the  bacilli  taken  directly  from  the  animal  body  grow 
only  very  imperfectly,  or,  in  many  cases,  not  at  all. 
From  this  it  seems  probable  that  there  is  a  difference 
in  the  nature  of  individual  tubercle  bacilli — some 
appearing  to  be  capable  of  growth  in  the  animal  tis- 
sues only,  while  others  are  apparently  possessed  of  the 
power  to  lead  a  limited  saprophytic  existence.  It  may 
be,  therefore,  that  those  bacilli  which  we  obtain  as  arti- 
ficial cultures  from  the  animal  body  are  offsprings  of 
the  more  saprophytic  varieties.  At  best,  one  never  sees 
with  the  tubercle  bacillus  a  saprophytic  condition  in 
any  way  comparable  to  that  possessed  by  many  of  the 
other  organisms  with  which  we  have  to  deal. 

For  the  cultivation  of  bacillus  tuberculosis  directly 
from  the  tissues  of  the  animal  the  method  by  which  one 
obtains  the  best  results  is  that  recommended  by  Koch, 
viz.,  cultivation  upon  blood-serum.  This  organism  is 
so  strictly  parasitic  that  even  very  slight  variations  in 
the  conditions  under  which  one  endeavors  to  isolate  it 
from  the  tissues  may  cause  total  failure.  It  is,  there- 
fore, necessary  that  the  injunctions  for  obtaining  it  in 
pure  culture  should  be  carefully  observed. 


324  BACTERIOLOGY. 

PREPARATION  OF  CULTURES  FROM  TISSUES. — Under 
strictest  antiseptic  precautions  remove  from  the  animal 
the  diseased  organ — the  liver,  spleen,  or  a  lymphatic 
gland  being  preferable.  Place  the  tissue  in  a  sterilized 
Petri  dish,  and  dissect  out  with  sterilized  scissors  and 
forceps  the  small  tubercular  nodules.  Place  each  nodule 
upon  the  surface  of  the  blood-serum,  one  nodule  in 
each  tube,  and  with  a  heavy,  sterilized,  looped  platinum 
needle  or  spatula  rub  it  carefully  over  the  surface.  It 
is  best  to  dissect  away  twenty  to  thirty  such  tubercles 
and  treat  each  in  the  same  way.  Some  of  the  tubes 
will  remain  sterile,  others  may  be  contaminated  by 
extraneous  saprophytic  organisms  during  the  manipula- 
tion, while  a  few  may  give  the  result  desired,  viz., 
a  growth  of  the  tubercle  bacillus  itself. 

The  blood-serum  upon  which  the  organism  is  to  be 
cultivated  should  be  comparatively  freshly  prepared — 
that  is,  should  not  be  dry. 

After  inoculating  the  tubes  they  should  be  carefully 
sealed  to  prevent  evaporation  and  consequent  dry- 
ing. This  is  done  by  burning  off  the  overhanging 
cotton  plug  in  a  gas-flame,  and  then  impregnating 
the  upper  layers  of  the  cotton  with  either  sealing- 
wax  or  paraffin  of  a  high  melting-point ;  or  by  insert- 
ing over  the  burned  end  of  the  cotton  plug  a  soft, 
closely  fitting  cork  that  has  been  sterilized  in  the 
steam  sterilizer  just  before  using  (Ghriskey).  This 
precaution  is  necessary  because  of  the  slow  growth  of 
the  organism.  Under  the  most  favorable  conditions 
tubercle  bacilli  directly  from  the  animal  body  show  no 
evidence  of  growth  for  about  twelve  days  after  inocu- 
lation upon  blood-serum,  and,  as  the/  must  be  retained 
during  this  time  at  the  body-temperature — 37.5°  C. — 


THE  TUBERCLE  BACILLUS.  325 

evaporation  would  take  place  very  rapidly  and  the 
medium  would  become  too  dry  for  their  development. 

If  these  primary  efforts  result  in  the  appearance  of  a 
culture  of  the  bacilli,  further  cultivations  may  be  made 
by  taking  up  a  bit  of  the  colony,  preferably  a  moder- 
ately large  quantity,  and  transferring  it  to  fresh  serum, 
and  this  in  turn  is  sealed  up  and  retained  at  the  same 
temperature.  Once  having  obtained  the  organism  in 
pure  culture,  its  subsequent  cultivation  may  be  con- 
ducted upon  the  glycerin-agar-agar  mixture — ordinary 
neutral  nutrient  agar-agar  to  which  from  4  to  6  per  cent, 
of  glycerin  has  been  added.  This  is  a  very  favorable 
medium  for  the  growth  of  this  organism  after  it  has 
accommodated  itself  to  its  saprophytic  mode  of  exist- 
ence, though  blood-serum  is  perhaps  the  best  medium 
to  be  employed  in  obtaining  the  first  generation  of  the 
organism  from  tuberculous  tissues. 

The  organism  may  be  cultivated  also  on  neutral  milk 
to  which  1  per  cent,  of  agar-agar  has  been  added,  also 
upon  the  surface  of  potato,  and  likewise  in  meat-infu- 
sion bouillon  containing  from  4  to  6  per  cent,  of 
glycerin. 

Cultures  of  the  tubercle  bacillus  are  characteristic 
in  appearance — once  having  seen  them  there  is  little 
probability  of  subsequent  mistake.  They  appear  as 
dry  masses,  which  may  develop  upon  the  surface  of 
the  medium  either  as  flat  scales  or  as  coarse  granular 
masses.  They  are  never  moist,  and  frequently  have  the 
appearance  of  dry  meal  spread  upon  the  surface  of  the 
medium.  In  the  lower  part  of  the  tube  in  which  they 
are  growing — i.  e.,  that  part  occupied  by  a  few  drops  of 
fluid  which  has  in  part  been  squeezed  from  the  medium 
during  the  process  of  solidification,  and  is  in  part  water 


326  BACTERIOLOGY. 

of  condensation — the  colonies  may  be  seen  to  float  as  a 
thin  pellicle  upon  the  surface  of  the  fluid. 

The  individuals  composing  the  growth  adhere  so 
tenaciously  together  that  it  is  with  the  greatest  diffi- 
culty they  can  be  separated.  In  even  the  oldest  and 
dryest  cultures  pulverization  is  impossible.  The  masses 
can  only  be  separated  and  broken  up  by  grinding  in 
a  mortar  with  the  addition  of  some  foreign  substance, 
such  as  very  fine,  sterilized  sand,  dust,  etc. 

The  cultures  are  of  a  dirty-drab  or  brownish-gray 
color  when  seen  on  serum  or  glycerin-agar-agar. 

On  potato  they  grow  in  practically  the  same  way, 
though  the  development  is  much  more  limited.  On 
this  medium  they  are  of  nearly  the  same  color  as  the 
potato  on  which  they  are  growing.  When  cultivated 
for  a  time  on  potato  they  are  said  to  lose  their  patho- 
genic properties. 

On  milk-agar-agar  they  are  of  so  nearly  the  same 
color  as  the  medium  that,  unless  they  are  growing  as 
characteristic  mealy-looking  masses,  considerably  ele- 
vated above  the  surface,  their  presence  is  less  conspicu- 
ous than  when  on  other  media. 

In  bouillon  they  grow  as  a  thin  pellicle  on  the  sur- 
face. This  may  fall  to  the  bottom  of  the  fluid  and  con- 
tinue to  develop,  its  place  on  the  surface  being  taken  by 
a  second  pellicle. 

The  tubercle  bacillus  does  not  develop  on  gelatin 
because  of  the  low  temperature  at  which  this  medium 
must  be  used. 

MICROSCOPIC  APPEARANCE  OF  BACILLUS  TUBER- 
CULOSIS.— Microscopically  the  organism  itself  is  a 
delicate  rod,  usually  somewhat  beaded  in  its  structure, 
though  rarely  it  is  seen  to  be  homogeneous.  It  is  either 


THE  TUBERCLE  BACILLUS.  327 

quite  straight,  or  somewhat  curved  or  bent  on  its  long 
axis.  In  some  preparations  involution-forms,  consisting 
of  rods  a  little  clubbed  at  one  extremity  or  slightly 
bulging  at  different  points,  may  be  detected.  Branch- 
ing forms  of  this  organism  have  been  described.  It 
varies  in  length — sometimes  being  seen  in  very  short 
segments,  again  much  longer,  though  never  as  long 
threads.  Usually  its  length  varies  from  2  to  5  p..  It 
is  commonly  described  as  being  in  length  about  one- 
fourth  to  one-half  the  diameter  of  a  red  blood-corpuscle. 
It  is  very  slender.  (See  Fig.  62.) 

These  rods  usually  present,  as  has  been  said,  an  ap- 
pearance of  alternate  stained  and  colorless  portions. 
The  latter  portions  are  believed  to  be  the  spores  of 
the  organism,  though  as  yet  no  incontestable  proof  of 
this  opinion  has  been  presented.  At  times  these  colorless 
portions  are  seen  to  bulge  slightly  beyond  the  contour 
of  the  rod,  and  in  this  way  give  to  the  rods  the  beaded 
appearance  so  commonly  ascribed  to  them. 

STAINING-PECULIARITIES. — A  peculiarity  of  this 
organism  is  its  behavior  toward  staining-reagents,  and 
by  this  means  alone  it  may  be  easily  recognized.  The 
tubercle  bacillus  does  not  stain  by  the  ordinary  methods. 
It  possesses  some  peculiarity  in  its  composition  that 
renders  it  proof  against  the  simpler  staining  processes. 
It  is  therefore  necessary  that  more  energetic  and  pene- 
trating reagents  than  the  ordinary  watery  solutions 
should  be  employed.  Experience  has  taught  us  that 
certain  substances  not  only  increase  the  solubility  of  the 
aniline  dyes,  but  by  their  presence  the  penetration  of 
the  coloring-agents  is  very  much  increased.  Two  of 
these  are  aniline  oil  and  carbolic  acid.  They  are 
employed  in  the  solutions  to  about  the  point  of  satura- 


328  BACTERIOLOGY. 

tion.     (For  the  exact  proportions,  see  chapter  on  Stain- 
ing-reagents.) 

Under  the  influence  of  heat  these  solutions  are  seen 
to  stain  all  bacteria  very  intensely — the  tubercle  bacilli 
as  well  as  other  forms.  If  we  subject  our  prepara- 
tion, which  may  contain  a  mixture  of  tubercle  bacilli 
and  other  species,  to  the  action  of  decolori zing-agents, 
another  peculiarity  of  the  tubercle  bacillus  will  be 
observed.  While  all  other  organisms  in  the  prepara- 
tion give  up  their  color  and  become  invisible,  the 
tubercle  bacillus  retains  it  with  marked  tenacity.  It 
stains  with  great  difficulty  ;  but  once  stained  it  retains 
the  color  even  under  the  action  of  strong  decolor- 
izing-agents. 

ORGANISMS   WITH   WHICH   BACILLUS   TUBERCULOSIS 
MAY   BE   CONFUSED. 

DIFFERENTIAL  DIAGNOSIS. — While  its  peculiar 
micro-chemical  reaction  is  usually  considered  to  be 
diagnostic  of  bacillus  tuberculosis,  it  is  well  to  re- 
member that  there  are  at  least  three  other  species 
of  bacilli  which,  when  similarly  treated,  react  in  the 
same  way.  It  is  of  importance  to  bear  this  point  in 
mind,  particularly  in  the  microscopic  examination  of 
urine  and  pathological  secretions  from  the  genito- 
urinary tract  and  from  the  rectum,  for  of  the  three 
species  two  are  frequently  found  in  these  localities, 
viz.,  the  so-called  smegma  bacillus,  located  in  the 
smegma  and  often  seen  beneath  the  prepuce  and  upon 
the  vulva,  both  normally  and  in  disease,  and  the  so- 
called  bacillus  of  syphilis,  described  by  Lustgarten  as 
contained  in  syphilitic  manifestations,  particularly  in 
primary  sores.  The  third  organism  of  this  group — the 


DIFFERENTIAL  DIAGNOSIS.  329 

bacillus  of  leprosy — because  of  its  rarity  is  not  so  likely 
to  cause  error  in  the  diagnosis  of  pathological  conditions 
occurring  in  these  localities. 

According  to  Hueppe,  the  differential  diagnosis  be- 
tween the  four  organisms  depends  upon  the  following 
reactions :  when  stained  by  the  carbol-fuchsin  method 
commonly  employed  in  staining  the  tubercle  bacillus 
the  syphilis  bacillus  becomes  almost  instantly  decolor- 
ized by  treatment  with  mineral  acids,  particularly  sul- 
phuric acid,  whereas  the  smegma  bacillus  resists  such 
treatment  for  a  much  longer  time,  and  the  lepra  and 
tubercle  bacillus  for  a  still  longer  time.  On  the  other 
hand,  if  decolorization  is  practised  with  alcohol,  instead 
of  acids,  the  smegma  bacillus  is  the  first  to  lose  its  color. 
The  bacillus  tuberculosis  and  the  bacillus  of  leprosy  are 
conspicuously  retentive  of  their  color  even  after  treat- 
ment with  both  acids  and  alcohol.  To  differentiate, 
then,  between  the  four  organisms  he  recommends  the 
following  order  of  procedure,  based  on  the  above  reac- 
tions : 

1.  Treat  the  preparation,  stained  with  carbol-fuchsin, 
with  dilute  sulphuric  acid  ;  the  syphilis  bacillus  becomes 
decolorized,  the  reaction  being  almost  instantaneous. 

2.  If  it  is  not  at  once  decolorized,  treat  with  alcohol ; 
if  it  is  the  smegma  bacillus,  this  will  rob  it  of  its  color. 

3.  If  it  is  still  not  decolorized,  it  is  either  the  lepra 
or  tubercle  bacillus. 

Grethe  recommends  *  the  following  as  a  trustworthy 
means  of  distinguishing  betweeil  the  tubercle  bacillus  and 
the  smegma  bacillus  :  stain  in  hot  carbol-fuchsin  solution, 
wash  in  water,  and  treat  the  preparation  with  a  saturated 
solution  of  methylene-blue  in  alcohol.  If  the  question- 
1  Fortschritte  der  Mediciii.,  1896,  Xo.  9. 


330  BACTERIOLOGY. 

able  organism  is  the  tubercle  bacillus,  it  retains  its  red 
color ;  if  the  smegma  bacillus,  the  red  color  is  dissolved 
by  the  alcohol  and  the  blue  color  is  substituted  for  it. 

The  differential  diagnosis  between  the  tubercle  bacil- 
lus and  the  lepra  bacillus  is  less  satisfactory ;  they  both 
take  the  same  stains,  and  both  retain  them  or  give 
them  up  under  treatment  with  the  same  decolorizers. 
The  results  of  investigations,  however,  indicate  differ- 
ences in  the  rate  of  staining  and  decolorization,  and  it  is 
stated  by  many  of  those  who  have  compared  the  two 
organisms  that  the  lepra  bacillus  takes  up  stain  very 
much  more  readily  than  does  the  tubercle  'bacillus, 
often  staining  perfectly  after  an  exposure  of  only  a  few 
minutes  to  cold  watery  solutions  of  the  dyes ;  but  when 
once  stained  it  retains  its  color  much  more  tenaciously 
when  acted  upon  by  decolorizing-agents  than  does  the 
latter  organism. 

According  to  Baumgarten,  the  lepra  bacillus  is  stained 
by  an  exposure  of  six  to  seven  minutes  to  a  cold,  satu- 
rated watery  solution  of  fuchsin,  and  retains  the  stain 
when  subsequently  treated  with  acid  alcohol  (nitric 
acid,  1  part;  alcohol,  10  parts).  By  similar  treatment 
for  the  same  length  of  time  bacillus  tuberculosis  does 
not  ordinarily  become  stained. 

These  points,  particularly  what  has  been  said  with  ref- 
erence to  the  smegma  bacillus  and  the  bacillus  of  syph- 
ilis, are  of  much  practical  importance,  and  should  always 
be  borne  in  mind  in  connection  with  the  microscopic 
examination  of  materials  to  which  these  organisms  are 
likely  to  gain  access.  It  is  hardly  necessary  to  say  that 
in  the  examination .  of  sputum  and  pathological  fluids 
from  other  parts  of  the  body  the  tubercle  bacillus  is, 
of  the  four  organisms,  always  the  one  most  commonly 


ANIMALS  SUSCEPTIBLE  TO  TUBERCULOSIS.    331 

encountered,  while  the  organism  described  by  Lust- 
garten  as  the  bacillus  of  syphilis  is  seen  so  rarely  that 
many  trustworthy  investigators  question  its  existence 
as  a  species  distinct  from  the  ordinary  smegma  bacillus. 

TUBERCULIN. — The  filtered  products  of  growth  from 
old  fluid  cultures  of  the  tubercle  bacillus  represent  what 
is  known  as  tuberculin — a  group  of  proteid  substances 
possessing  most  interesting  properties.  When  injected 
subcutaneously  into  healthy  subjects  tuberculin  has  no 
effect ;  but  when  introduced  into  the  body  of  a  tuber- 
culous person  or  animal  a  pronounced  systemic  reaction 
results,  consisting  of  sudden  but  temporary  elevation  of 
temperature,  with,  at  the  same  time,  the  occurrence 
of  marked  hypersemia  about  the  tuberculous  focus, 
a  change  histologically  analogous  to  that  seen  in  the 
primary  stages  of  acute  inflammation.  This  zone  of 
hypersemia,  with  the  coincident  exudation  and  infiltra- 
tion of  cellular  elements,  probably  aids  in  the  isolation 
or  casting  off  of  the  tuberculous  nodule,  the  inflamma- 
tory zone  forming,  so  to  speak,  a  line  of  demarcation 
between  the  diseased  and  healthy  tissue. 

As  a  curative  agent  for  the  treatment  of  tuberculosis, 
tuberculin  has  not  merited  the  confidence  that  wras  at 
first  accorded  to  it.  Its  field  of  usefulness  is  now 
almost  limited  to  the  diagnosis  of  obscure  cases,  and 
even  for  this  purpose  it  is  less  frequently  employed 
than  formerly. 

In  veterinary  medicine  it  has  proved  much  more 
trustworthy  as  a  diagnostic  aid,  and  is  practically 
everywhere  in  use  for  the  detection  of  incipient  tuber- 
culosis, especially  in  cattle. 

SUSCEPTIBILITY  OF  ANIMALS  TO  TUBERCULOSIS. — 


332  BACTERIOLOGY. 

The  animals  that  are  known  to  be  susceptible  to  tu- 
berculosis are  man,  apes,  cattle,  horses,  sheep,  guinea- 
pigs,  pigeons,  rabbits,  cats,  and  field-mice.  White  mice, 
dogs,  and  rats  possess  immunity  from  the  disease. 

We  have  reviewed  the  three  common  pathogenic 
organisms  with  which  we  may  come  in  contact  in  the 
sputum  of  tuberculous  individuals.  Occasionally  other 
forms  may  be  present.  The  pyogenic  forms  are  not 
rarely  found,  and  for  some  time  after  an  attack  of 
diphtheria  the  bacillus  of  Loffler  is  demonstrable  in 
the  pharynx,  so  that  it,  too,  may  be  present  under 
exceptional  circumstances. 

From  time  to  time  fowls  are  known  to  suffer  from 
a  form  of  tuberculosis  that  in  a  number  of  ways  sug- 
gests human  or  mammalian  tuberculosis.  The  bacillus 
causing  the  disease,  the  so-called  bacillus  of  fowl  tuber- 
culosis, bacillus  tuberculosis  avium,  while  simulating  the 
genuine  bacillus  tuberculosis  morphologically,  diifers 
from  it  both  in  cultural  and  pathogenic  peculiarities. 
Thus,  for  instance,  it  develops  into  much  longer  and 
somewhat  thinner  threads ;  grows  rapidly  on  media 
without  glycerin  or  glucose  ;  does  not  grow  on  potato ; 
develops  as  well  at  from  42°  to  43°  C.  as  at  37°  to  38° 
C.  ;*  its  virulence  is  not  diminished  by  cultivation  at 
43°  C. ;  development  on  artificial  media  begins  in  from 
six  to  eight  days  after  inoculation ;  young  cultures  on 
solid  media  are  whitish,  soft,  and  moist,  becoming  yel- 
lowish and  slimy  with  age ;  it  is  somewhat  more  resist- 
ant to  drying  and  high  temperatures  than  the  bacillus 
of  mammalian  tuberculosis ;  the  results  of  its  patho- 

1  The  normal  body-temperature  of  fowls  ranges  between  41.5°  and 
42.5°  C. 


ANIMALS  SUSCEPTIBLE  TO   TUBERCULOSIS.   333 

genie  activities  are  almost  always  chronic,  are  rarely 
located  in  the  lungs  or  intestines,  but  are  especially  fre- 
quent in  the  liver  and  spleen ;  the  lesions  are  conspic- 
uously rich  in  bacilli,  do  not  show  the  central  necrotic 
area  that  characterizes  the  mammalian  tubercle;  the 
disease  is  transmissible  from  the  hen  to  the  embryo 
chick ;  the  only  susceptible  mammal  is  the  rabbit ;  the 
guinea-pig  and  dog  are  naturally  immune ;  it  has  the 
same  micro-chemical  staining-reactions  as  mammalian 
bacillus  tuberculosis;  it  has  never  been  certainly  de- 
tected in  human  tuberculosis. 

Some  are  inclined  to  regard  this  organism  as  but  a 
variety  of  the  genuine  bacillus  tuberculosis,  and  it  is  not 
unreasonable  to  believe  that  the  sojourn  of  that  organ- 
ism in  the  body  of  a  refractory  animal,  whose  normal 
temperature  is  so  high  as  that  of  the  fowl,  when  not  fatal 
to  the  organism,  might  result  in  striking  modifications 
of  certain  of  its  biological  functions.  In  fact,  Nocard  * 
has  shown  that  if  the  genuine  bacillus  tuberculosis  from 
man  be  left  in  the  peritoneal  cavity  of  chickens  (by  the 
collodion-sac  method  of  Metschnikoff,  Roux,  and  Sal- 
lembini,  which  see)  for  from  five  to  eighth  months,  they 
will,  by  the  end  of  this  time,  have  become  so  modified 
in  their  biological  peculiarities  as  to  simulate  very 
closely  the  bacillus  of  fowl  tuberculosis. 

Theobald  Smith 2  has  called  attention  to  certain  very 
conspicuous  differences  that  may  be  observed  between 
the  bacilli  obtained  from  human  and  those  from  bovine 
tuberculosis. 

Anatomical  lesions  very  suggestive  of,  though  not 

1  Nocard :  Annales  de  1'Institut  Pasteur,  1898,  p.  561. 
*  Smith :   Transactions  Association   of  American   Physicians,  1896, 
vol.  xi.  p.  75. 


334  BACTERIOLOGY. 

identical  with,  those  produced  by  bacillus  tuberculosis 
have  also  from  time  to  time  been  observed  in  mice,  rats, 
guinea-pigs,  rabbits,  cats,  goats,  bovines,  hogs,  and  man. 
They  do  not  appear  to  be  of  a  specific  nature  as  regards 
etiology,  for  the  reason  that  different  authors  have  de- 
scribed different  species  of  bacilli  as  the  causative 
agents.  These  conditions  suggest  tuberculosis  only  by 
the  more  superficial  character  of  the  lesions,  for  in  no 
instance  have  the  organisms  detected  been  in  any  way 
similar  to  the  genuine  bacillus  tuberculosis.  These  affec- 
tions usually  pass  under  the  name  pseudo-tuberculosis. 

THE    BACILLUS   OF    INFLUENZA. 

An  important  historic  epidemic  disease,  on  the  nature 
of  which  much  light  has  been  shed  through  modern 
methods  of  investigation,  is  influenza.  Quoting  Hirsch, 
the  first  trustworthy  literary  records  that  we  have  of 
this  disease  date  from  the  early  part  of  the  twelfth 
century. 

Between  1173  and  1874  it  made  its  epidemic  or  pan- 
demic appearance  on  eighty-six  different  occasions.  Its 
first  appearance  in  this  country  was  in  Massachusetts  in 
1627  ;  since  that  time  there  have  been  twenty -two  vis- 
itations of  influenza  to  the  United  States.  The  pan- 
demic of  1889-790,  the  most  severe  for  a  long  time, 
appears  to  have  originated  in  Central  Asia  and  to  have 
spread  pretty  much  over  the  entire  civilized  world.  The 
advent  of  influenza  in  a  community  is  always  remark- 
able for  its  astonishing  rate  of  transmission  from  per- 
son to  person  and  its  dissemination  over  wide  areas. 

During  the  recent  pandemic  investigations  having 
for  their  object  the  discovery  of  its  cause  were  insti- 
tuted, with  the  result  of  demonstrating  in  the  catarrhal 


THE  BACILLUS  OF  INFLUENZA.  335 

secretions  from  the  air-passages  a  micro-organism  that 
is  claimed  to  stand  in  causal  relation  to  influenza*. 

This  organism,  bacillus  influenzas,  as  it  is  called,  was 
discovered,  isolated,  cultivated,  and  described  by  R. 
Pfeiffer. 

It  is  a  very  small,  slender,  non-spore-forming,  non- 
motile,  aerobic  bacillus,  occurring  singly  and  in  pairs, 
joined  end  to  end.  It  stains  with  watery  solutions  of 
the  ordinary  basic  aniline  dyes ;  somewhat  better  with 
alkaline  methylene-blue,  but  best  when  treated  for  five 
minutes  with  a  dilution  of  ZiehPs  carbol-fuchsin  in 
water  (the  color  of  the  solution  should  be  pale  red). 
(Fig.  64.)  It  is  decolorized  by  the  method  of  Gram. 

FIG.  64. 


Bacillus  of  influenza  in  sputum. 


It  develops  only  at  temperatures  ranging  from  26° 
to  43°  C.  Its  optimum  temperature  for  growth  is 
37°  C.  It  possesses  the  peculiarity  of  developing  upon 
only  those  artificial  culture-media  to  which  blood  or 


336  BACTERIOLOGY. 

blood-coloring-matter  has  been  added.  Its  cultivation 
is  best  conducted  and  its  development  most  satisfac- 
torily observed  by  the  following  procedure :  over  the 
surface  of  a  slanted  agar  tube  or  over  agar-agar  solid- 
ified in  a  Petri  dish  smear  a  small  quantity  of  sterile 
blood  (not  blood-serum).  A  bit  of  the  mucus  from  the 
sputum  of  the  influenza  patient  is  then  taken  up  with 
sterilized  forceps  or  on  a  sterilized  wire  loop,  rinsed 
in  sterile  bouillon  or  water  and  rubbed  over  the  sur- 
face of  the  prepared  agar-agar.  The  plate  or  tube  is 
then  placed  in  the  incubator  at  37°  to  38°  C.  If  in- 
fluenza bacilli  be  present,  they  will  develop  as  minute, 
transparent,  watery  colonies  that  are  without  structure, 
and  which  resemble  somewhat  minute  drops  of  dew. 
They  are  discrete  and  show  little  or  no  tendency  to 
coalesce. 

If  a  small  bit  of  mucus  be  rubbed  over  the  surface 
of  ordinary  nutrient  agar-agar,  no  such  colonies  de- 
velop. In  making  the  diagnosis  by  this  method  cult- 
ures on  both  agar-agar  containing  blood  (not  blood- 
serum)  and  agar-agar  containing  no  blood  should  always 
be  made,  for  the  reason  that  growth  of  these  peculiar 
colonies  in  the  former  and  no  such  growth  in  the  latter 
are  evidence  that  one  is  dealing  with  materials  from  a 
case  of  influenza. 

The  organism  may  also  be  cultivated  in  bouillon  to 
which  blood  has  been  added,  if  kept  at  body-tempera- 
ture. The  growth  appears  as  whitish  flakes.  Since  this 
organism  is  a  strict  aerobe,  its  cultivation  can  only  be 
conducted  on  the  surface  of  the  medium  used — i.  e., 
where  it  has  freest  access  to  oxygen.  It  is  therefore 
inadvisable  to  prepare  plates  in  the  usual  way.  When 
its  cultivation  is  attempted  in  bouillon  it  is  recom- 


THE  BACILLUS  OF  INFLUENZA.  337 

mended,  in  order  to  favor  the  free  diffusion  of  oxygen, 
that  the  depth  of  fluid  be  very  shallow. 

Contrary  to  what  might  be  supposed,  the  bacillus  of 
influenza  has  very  little  tenacity  of  life  outside  of  the 
diseased  body.  It  is  destroyed  in  from  two  to  three 
h<mrs  by  rapid  drying,  and  in  from  eight  to  twenty- 
four  hours  when  dried  more  slowly.  Cultures  retain 
their  vitality  for  from  two  to  three  weeks.  The  organ- 
ism dies  in  water  in  a  little  over  a  day.  As  a  result  of 
these  observations,  Pfeiffer  does  not  believe  the  disease 
to  be  disseminated  by  either  the  air  or  the  water,  but 
rather  by  direct  infection  from  the  catarrhal  secretions 
of  the  patients. 

This  organism  has  not  been  found  outside  of  the 
human  body.  In  the  influenza  patient  it  is  present  in 
the  catarrhal  secretions,  bronchial  mucous  membrane, 
and  the  diseased  lung-tissues.  It  may  be  demonstrated 
microscopically  in  the  mucus  by  cover-slip  prepara- 
tions made  in  the  usual  way  and  stained  with  diluted 
carbol-fuchsin,  referred  to  above.  In  the  tissues  it 
may  be  demonstrated  in  sections  stained  in  the  same 
solution.  In  the  sputum  the  bacilli  are  found  as  masses 
and  as  scattered  cells.  (See  Fig.  64.)  They  are  also 
found  writhin  the  bodies  of  leucocytes,  especially  in  the 
later  stages  of  the  disease  when  convalescence  has  set 
in ;  at  this  time  they  appear  as  very  small,  irregular, 
evidently  degenerated  bacilli  within  white  blood- 
corpuscles.  They  are  also  present  in  the  nasal  secre- 
tions. 

At  autopsies  it  is  advisable  to  cut  out  pieces  of 
the  diseased  tissue  about  the  size  of  a  pea  or  a  bean, 
break  them  up  in  a  small  quantity  of  sterile  water  or 
bouillon,  and  make  the  cultures  from  this  infusion. 

22 


338  BACTERIOLOGY. 

By  this  procedure  two  advantages  are  gained :  first, 
a  dilution  of  the  number  of  bacteria  present;  and, 
secondly,  the  tissue  furnishes  the  amount  of  haemo- 
globin necessary  for  the  growth  of  the  organism. 
Under  these  circumstances  it  is,  of  course,  not  neces- 
sary to  make  a  further  addition  of  blood  to  the  culture- 
medium. 

The  only  animal  that  has  been  found  susceptible 
to  inoculation  with  this  organism  is  the  monkey.  By 
intratracheal  injection  Pfeiffer  succeeded  in  causing  a 
toxic  condition  that  proved  fatal.  He  does  not  regard 
the  death  of  the  animals  as  due  to  infection,  but  rather 
to  intoxication.  The  disease,  as  seen  in  man,  has  not 
been  reproduced  in  animals. 


CHAPTER  XIX. 

Glanders — Characteristics  of  the  disease — Histological  structure  of  the 
glanders  nodule — Susceptibility  of  different  animals  to  glanders — 
The  bacillus  of  glanders ;  its  morphological  and  cultural  peculiari- 
ties— Diagnosis  of  glanders. 

SYNONYMS  :  Rotz  (Ger.),  Morve  (Fr.). 

The  disease  is  generally  known  as  glanders  when  the 
mucous  membrane  of  the  nostrils  is  affected,  and  as  farcy 
when  the  subcutaneous  lymphatics  are  the  principal  sites 
of  involvement. 

Though  most  commonly  seen  in  the  horse  and  ass, 
glanders  is  not  rarely  met  with  in  other  animals,  and  is 
occasionally  encountered  in  man.  When  occurring 
spontaneously  in  the  horse  its  primary  seat  is  usually 
upon  the  mucous  membrane  of  the  nostrils.  It  appears 
in  the  form  of  small  gray  nodules,  about  which  the 
membrane  is  congested  and  swollen.  These  nodules 
ultimately  coalesce  to  form  ulcers.  There  is  a  profuse 
slimy  discharge  from  the  nostrils  during  the  course  of  the 
disease.  The  primary  lesion  may  extend  from  its  seat  in 
the  nose  to  the  mouth,  larynx,  trachea,  and  ultimately 
to  the  lungs.  Its  secondary  manifestations  are  observed 
along  the  lymphatics  that  communicate  with  the  initial 
focus ;  in  the  lymphatic  glands,  and  as  metastatic  foci 
in  the  internal  organs.  Less  frequently  the  disease  is 
seen  to  begin  in  the  skin,  particularly  in  the  region  of 
the  neck  and  breast.  When  in  this  locality  the  sub- 
cutaneous lymphatics  become  involved,  and  are  con- 

339 


340  BACTERIOLOGY. 

verted  into  indurated,  knotty  cords — "farcy-buds" — 
easily  discernible  from  without. 

In  man  it  usually  occurs  in  individuals  who  have 
been  in  attendance  upon  animals  affected  with  the  dis- 
ease. It  may  occur  upon  the  mucous  membrane  of  the 
nares ;  but  its  most  frequent  expressions  are  in  the  skin 
and  muscles,  where  appear  abscesses,  phlegmons,  ery- 
sipelas-like inflammations,  and  local  necrosis  closely 
resembling  carbuncles.  Metastases  to  the  lungs,  kid- 
neys, and  testicles,  as  in  the  horse,  may  also  be  seen. 

When  occurring  upon  the  mucous  membrane  glan- 
ders is  characterized  by  the  presence  of  gray  nod- 
ules, about  as  large  as  a  pin-head,  that  closely  re- 
semble miliary  tubercles  in  their  naked-eye  appearance. 
These  consist  histologically  of  granulation-tissue — i.  e., 
of  small  round  cells,  very  similar  to  proliferating 
leucocytes — of  some  lymph-cells,  and,  in  the  earliest 
stages,  of  a  small  portion  of  necrotic  tissue.  As  they 
grow  older,  and  the  process  advances,  there  is  a 
tendency  to  central  necrosis,  with  the  ultimate  for- 
mation of  a  soft,  yellow,  creamy,  pus-like  material. 
Though  strikingly  like  miliary  tubercles  in  certain 
respects  in  the  early  stages,  they  present,  nevertheless, 
decided  points  of  difference  when  examined  more  in 
detail. 

The  round-cell  infiltration  of  the  glanders  nodule 
consists  essentially  of  polynuclear  leucocytes,  while  that 
of  the  miliary  tubercle  partakes  more  of  the  nature  of  a 
lymphocytic  infiltration  ;  in  the  later  stages  of  the  proc- 
ess the  glanders  nodule  breaks  down  into  a  soft  creamy 
matter,  very  analogous  to  ordinary  pus,  while  in  the 
later  stages  of  the  miliary  tubercle  the  tendency  is 
to  an  amalgamation  of  its  histological  constituents, 


THE  BACILLUS  OF  GLANDERS.  341 

and  ultimately  to  necrosis  with  caseation.  The  giant- 
cell  formation  common  to  tuberculosis  is  never  seen  in 
the  glanders  nodule.  As  Baumgarten  aptly  puts  it: 
"  The  pathological  manifestations  of  glanders,  from  the 
histological  aspect,  stand  midway  between  the  acute 
purulent  and  the  chronic  inflammatory  processes." l 
Evidently  these  differences  are  only  to  be  explained 
by  differences  in  the  nature  of  the  causes  that  underlie 
the  several  affections.  We  have  studied  the  character- 
istics of  bacillus  tuberculosis ;  we  shall  now  take  up  the 
bacillus  of  glanders  and  note  the  striking  differences 
between  them. 

THE  BACILLUS  OF  GLANDERS  (BACILLUS   MALLEl). 

In  1882  Loffler  and  Schiitz  discovered  in  the  diseased 
tissues  of  animals  suffering  from  glanders  a  bacillus  that, 
when  isolated  in  pure  culture  and  inoculated  into  sus- 
ceptible animals,  possesses  the  property  of  reproducing 
the  disease  with  all  its  clinical  and  pathological  mani- 
festations. It  is  therefore  the  cause  of  the  disease. 

This  organism  is  a  rod,  with  rounded  or  slightly 
pointed  ends.  It  usually  stains  somewhat  irregularly. 
(See  Fig.  65.)  When  examined  in  stained  prepara- 
tions its  continuity  is  marked  by  alternating  darkly 
and  lightly  stained  areas.  It  is  usually  seen  as  a 
single  rod,  but  may  occur  in  pairs,  and  less  frequently 
in  longer  filaments. 

The  question  as  to  its  spore-forming  property  is  still 
an  open  one,  though  the  weight  of  evidence  is  in  oppo- 

1  For  a  further  discussion  of  the  pathology  and  pathogenesis  of  this 
disease,  see  Lehrbuch  der  pathologischen  Mykologie,  by  Baumgarten, 
1890.  See,  also,  Wright :  "  The  Histological  Lesions  of  Acute  Glanders 
in  Man,"  Journal  of  Experimental  Medicine,  vol.  i.  p.  577. 


342 


BACTERIOLOGY. 


sition  to  the  opinion  that  it  possesses  this  peculiarity. 
Certain  observers  claim  to  have  demonstrated  spores  in 
the  bacilli  by  particular  methods  of  staining;  but  this 
statement  can  have  but  little  weight  when  compared 
with  the  behavior  of  the  organism  when  subjected  to 
more  conclusive  tests.  For  example,  it  does  not,  at 
any  stage  of  development,  resist  exposure  to  3  per  cent. 

FIG.  65. 


Bacillus  of  glanders  (bacillus  mallei),  from  culture. 

carbolic  acid  solution  for  longer  than  five  minutes,  nor 
to  1  :  5000  sublimate  solution  for  more  than  two  min- 
utes. It  is  destroyed  in  ten  minutes  in  some  experi- 
ments, and  in  five  in  others,  by  a  temperature  of  55°  C. ; 
and  when  dried  it  loses  its  vitality,  according  to  dif- 
ferent observers,  in  from  thirty  to  forty  days ;  all  of 
which  speak  directly  against  this  being  a  spore-bearing 
bacillus. 

It    is  not   motile,   and    does   not   therefore    possess 
flagella. 


THE  BACILLUS  OF  GLANDERS.  343 

It  grows  readily  on  ordinary  nutrient  media  at  from 
25°  to  38°  C. 

Upon  nutrient  agar-agar,  both  with  and  without 
glycerin,  it  appears  as  a  moist,  opaque,  glazed  layer, 
with  nothing  characteristic  about  it.  This  is  true  both 
for  smear-cultures  and  for  single  colonies. 

Its  growth  on  gelatin  is  much  less  voluminous  than 
on  media  that  can  be  kept  at  higher  temperature,  though 
it  does  grow  on  this  media  at  room-temperature  without 
causing  liquefaction. 

Its  growth  on  blood-serum  is  in  the  form  of  a 
moist,  opaque,  slimy  layer,  inclining  to  a  yellowish  or 
dirty,  brownish-yellow  tinge.  It  does  not  liquefy  the 
serum. 

On  potato  its  growth  is  moderately  rapid,  appearing 
in  from  twenty-four  to  thirty-six  hours  at  37°  C.  as  a 
moist,  amber-yellow,  transparent  deposit  having  some- 
what the  appearance  of  honey;  this  becomes  deeper 
in  color  and  denser  in  consistence  as  growth,  progresses, 
and  finally  takes  on  a  reddish-brown  color ;  at  the  same 
time  the  potato  about  it  becomes  darkened. 

In  bouillon  it  causes  diffuse  clouding,  with  ultimately 
the  formation  of  a  more  or  less  tenacious  or  ropy  sedi- 
ment. 

In  milk  to  which  a  little  litmus  has  been  added  it 
causes  the  blue  color  to  become  red  or  reddish  in  from 
four  to  five  days,  and  quite  red  after  two  weeks  at  37°  C. 
At  the  same  time  the  milk  separates  into  clear  whey 
and  a  firm  clot  of  casein. 

Its  reactions  to  heat  are  very  interesting.  At  42°  C. 
it  will  often  grow  for  twenty  days  or  more.  It  will 
not  grow  at  43°  C.,  and  if  exposed  to  this  temperature 
for  forty-eight  hours  it  is  destroyed.  It  is  killed  in  five 


344  BACTERIOLOGY. 

hours  when  exposed  to  50°  C.,  and  in  five  minutes  by 
55°  C. 

It  grows  both  with  and  without  oxygen ;  it  is  there- 
fore facultative  as  regards  its  relation  to  this  gas. 

On  cover-slips  it  stains  readily  with  all  the  basic 
aniline  dyes,  and,  as  a  rule,  as  stated,  presents  conspic- 
uous irregularities  in  the  way  that  it  takes  up  the  dyes, 
being  usually  marked  by  deeply  stained  areas  that  alter- 
nate with  points  at  which  it  either  does  not  stain  at  all 
or  only  slightly. 

The  animals  susceptible  to  infection  by  this  organism 
are  horses,  asses,  field-mice,  guinea-pigs,  and  cats. 
Baurngarten  records  cases  of  infection  in  lions  and 
tigers  that  were  fed,  in  menageries,  with  flesh  from 
horses  aifected  with  the  disease.  Rabbits  are  but 
slightly  susceptible  ;  dogs  and  sheep  still  less  so.  Man 
is  susceptible,  and  infection  not  rarely  terminates  fatally. 
White  mice,  common  gray  house-mice,  rats,  cattle,  and 
hogs  are  insusceptible. 

INOCULATION  EXPERIMENTS. — The  most  favorable 
animal  upon  which  to  study  the  pathogenic  properties 
of  this  organism  in  the  laboratory  is  the  common  field- 
mouse.  When  inoculated  subcutaneously  with  a  small 
portion  of  a  pure  culture  of  the  glanders  bacillus  death 
ensues  in  about  seventy-two  hours.  The  most  conspicu- 
ous tissue-changes  will  be  enlargement  of  the  spleen, 
which  is  at  the  same  time,  almost  constantly,  studded 
with  minute  gray  nodules,  the  typical  glanders  nodule. 
They  are  rarely  present  in  the  lungs,  but  may  frequently 
be  seen  in  the  liver.  From  these  nodules  the  glanders 
bacillus  may  be  obtained  in  pure  culture.  With  the 
exception  of  the  characteristic  nodules,  the  disease  as 
seen  in  this  animal  presents  none  of  the  features  that 


GLANDERS  BACILLUS.  345 

it  displays  in  the  horse  and  ass.  The  clinical  and.  path- 
ological manifestations  resulting  from  inoculation  of 
guinea-pigs  are  much  more  faithful  reproductions.  The 
animal  lives  usually  from  six  to  eight  weeks  after  inocu- 
lation, and  during  this  time  becomes  affected  with  a  group 
of  most  interesting  and  peculiar  pathological  processes. 
The  specific  inflammatory  condition  of  the  mucous 
membrane  of  the  nostrils  is  almost  always  present.  The 
joints  become  swollen  and  infiltrated  to  such  an  extent 
as  often  to  interfere  with  the  use  of  the  legs.  In  male 
animals  the  testicles  become  enormously  distended  with 
pus,  and  on  closer  examination  a  true  orchitis  and  epi- 
didymitis  are  seen  to  be  present.  The  internal  organs, 
particularly  the  lungs,  kidneys,  spleen,  and  liver,  are 
usually  the  seat  of  the  nodular  formations  characteristic 
of  the  disease.  From  all  of  these  disease-foci  the 
bacillus  causing  them  can  be  isolated  in  pure  culture. 

STAINING  IN  TISSUES. — Though  always  present  in 
the  diseased  tissues,  considerable  trouble  is  usually 
experienced  in  demonstrating  the  bacteria  by  staining- 
methods.  The  difficulty  is  due  to  the  fact  that  the  bacilli 
are  very  easily  decolorized,  and  in  tissues  stained  by 
the  ordinary  processes '  are  robbed  of  their  color  even 
by  the  alcohol  with  which  the  tissue  is  rinsed  and  de- 
hydrated. If  we  will  remember  not  to  employ  con- 
centrated stains,  and  not  to  expose  the  sections  to  the 
stains  for  too  long  a  time,  but  little  treatment  with 
decolorizing-agents  is  necessary,  and  very  satisfactory 
preparations  will  be  obtained.  A  number  of  methods 
have  been  suggested  for  staining  the  glanders  bacilli 
in  tissues,  and  if  what  has  been  said  will  be  borne  in 
mind,  no  difficulty  should  be  experienced. 

Two  satisfactory  methods  that  we  have  used  for  this 


346  BACTERIOLOGY. 

purpose,  though  perhaps  no  better  than  some  of  the 
others,  are  as  follows  : 

a.  Transfer   the   sections    from   alcohol  to  distilled 
water.     This  lessens  the  intensity  with  which  the  stain 
subsequently  takes   hold   of  the  tissues,  by  diminish- 
ing the  activity'  of  the  diffusion  that  would  occur  if 
they  were  placed  from  alcohol  into  watery  solutions  of 
the  dyes.     Transfer  from  distilled  water  to  the  slide, 
absorb   all  water  with  blotting-paper,  and   stain  with 
two  or  three  drops  of 

Carbol-fuchsin  .    .   : 10  c.c. 

Distilled  water 100  c.c. 

for  thirty  minutes ;  absorb  all  superfluous  stain  with 
blotting-paper,  and  wash  the  section  three  times  with 
0.3  per  cent,  acetic  acid,  not  allowing  the  acid  to  act 
for  more  than  ten  seconds  each  time.  Remove  all 
acid  from  the  section  by  carefully  washing  in  distilled 
water ;  absorb  all  water  by  gentle  pressure  with  blot- 
ting-paper ;  and  finally,  at  very  moderate  heat,  or  with 
a  small  bellows  (Kiihne),  dry  the  section  completely  on 
the  slide.  When  dried  clear  in  xylol  and  mount  in 
xylol  balsam. 

b.  Transfer  the   sections   from   alcohol   to   distilled 
water;  from  water  to  the  dilute  fuchsin  solution,  and 
gently    warm   (about    50°    C.)   for    fifteen   to   twenty 
minutes.     Transfer  sections  from  the  staining-solution 
to  the  slide,  absorb  all  superfluous  stain  with  blotting- 
paper,  and  then  treat  them  with  1  per  cent,  acetic  acid 
from  one-half  to  three-quarters  of  a  minute.     Remove 
all  trace  of  acid  with  distilled  water,  absorb  all  water  by 
gentle  pressure  with  blotting-paper,  and  then  treat  the 
sections  with  absolute   alcohol  by  allowing   it  to  flow 


DIAGNOSIS  BY  METHOD  OF  STRAUSS.         347 

over  them  drop  by  drop.  For  small  sections  three  or 
four  drops  are  sufficient.  Under  no  circumstances 
should  the  alcohol  be  allowed  to  act  for  more  than 
one-quarter  of  a  minute.  Clear  in  xylol  and  mount 
in  xylol  balsam. 

By  method  6  the  tissues  are  better  preserved  than  by 
method  a,  by  which  they  are  dried. 

Very  good  preparations  are  also  obtained  by  the  use 
of  Loffler's  alkaline  methylene-blue,  if  care  be  taken 
not  to  stain  for  too  long  a  time  nor  to  decolorize  too 
energetically  with  alcohol. 

No  method  of  contrast-stain  for  this  organism  in 
tissues  has  been  devised. 

In  properly  stained  tissues  the  bacilli  will  be  found 
most  numerous  in  the  centre  of  the  nodules,  becoming 
fewer  as  we  approach  the  periphery.  They  usually  lie 
between  the  cells,  but  at  times  may  be  seen  almost 
filling  some  of  the  epithelial  cells,  of  which  the  nodule 
contains  more  or  less.  They  are  always  present  in 
these  nodules  in  the  tissues;  they  are  rarely  present 
in  the  blood,  and,  if  so,  in  only  small  numbers. 

DIAGNOSIS  OF  THE  DISEASE  BY  THE  METHOD  OF 
STRAUSS. — From  what  has  been  said,  the  diagnosis  of 
glanders  by  routine  bacteriological  methods  is  certain 
and  relatively  easy,  but  requires  time.  In  clinical  work 
it  is  of  great  importance  for  the  diagnosis  to  be  estab- 
lished as  quickly  as  possible.  With  this  in  view  Strauss 
devised  a  method  that  has  given  entirely  satisfactory 
results.  It  consists  in  introducing  into  the  peritoneal 
cavity  of  a  male  guinea-pig  a  bit  of  the  suspected  tissue 
or  culture.  If  it  be  from  a  genuine  case  of  glanders, 
the  testicles  begin  to  swell  in  about  thirty  hours,  and 
as  this  proceeds  the  skin  over  them  becomes  red  and 


348  BACTERIOLOGY. 

shining,  desquamation  occurs,  evidences  of  pus-forma- 
tion are  seen,  and,  indeed,  the  abscess  (purulent  orchitis) 
often  breaks  through  the  skin.  The  diagnostic  sign  is 
the  tumefaction  of  the  testicles. 

MALLEIN. — The  filtered  products  of  growth  of  the 
glanders  bacillus  in  fluid  media  represent  what  is  known 
as  mallein — a  group  of  compounds  that  bear  to  glanders 
pretty  much  the  same  relation  that  tuberculin  bears  to 
tuberculosis.  It  is  used  with  considerable  success  as  a 
diagnostic  aid  in  detecting  the  existence  or  absence  of 
deep-seated  manifestations  of  the  disease,  the  glanderous 
animal  reacting  to  subcutaneous  injections  of  mallein  in 
from  four  to  ten  hours,  while  an  animal  not  so  affected 
gives  no  such  reactions. 

Mallein  is  prepared  from  old  glycerin-bouillon  cult- 
ures of  the  glanders  bacillus  by  steaming  them  for 
several  hours  in  the  sterilizer,  after  which  they  are 
filtered  through  unglazed  porcelain. 


CHAPTER  XX. 

Bacillus  diphtherix — Its  isolation  and  cultivation — Morphological  and 
cultural  peculiarities — Pathogenic  properties — Variations  in  viru- 
lence— Pseudo-diphtheria  bacilli. 

FROM  the  gray-white  deposit  on  the  fauces  of  a  diph- 
theritic patient  prepare  a  series  of  cultures  in  the  fol- 
lowing way : 

Have  at  hand  five  or  six  tubes  of  Loffler's  blood- 
serum  mixture.  (See  chapter  on  Media.)  Pass  a  stout 
platinum  needle,  which  has  been  sterilized,  into  the 
membrane  and  twist  it  around  once  or  twice,  or  brush 
it  gently  over  the  surface  of  the  membrane.  Without 
touching  it  against  anything  else  rub  it  carefully  over 
the  surface  of  one  of  the  serum-tubes ;  without  steriliz- 
ing it  pass  it  over  the  surface  of  the  second,  then  the 
third,  fourth,  and  fifth  tubes.  Place  these  tubes  in  the 
incubator.  Then  prepare  cover-slips  from  scrapings 
from  the  membrane  on  the  fauces.  If  the  case  is  one 
of  true  diphtheria,  the  tubes  will  be  ready  for  examina- 
tion on  the  following  day. 

The  reason  that  plates  are  not  made  in  the  regular 
way  in  this  examination  is  that  the  bacillus  of  diph- 
theria develops  much  more  luxuriantly  on  the  serum 
mixture,  from  which  plates  cannot  be  made,  than  it 
does  on  the  media  from  which  they  can  be  made.  The 
method  employed,  however,  insures  a  dilution  in  the 
number  of  organisms  present,  and  this,  in  addition  to 
the  fact  that  the  blood-serum  mixture  is  a  much  more 

349 


350  BACTERIOLOGY. 

favorable  medium  for  the  rapid  development  of  the 
diphtheria  bacillus  than  of  the  other  organisms  present, 
makes  its  isolation  by  this  method  a  matter  of  but  little 
difficulty. 

After  twenty-four  hours  in  the  incubator  the  tubes 
present  a  characteristic  appearance.  Their  surfaces  are 
marked  by  more  or  less  irregular  patches  of  a  white 
or  cream-colored  growth,  which  is  usually  more  dense 
at  the  centre  than  at  the  periphery.  Except  now  and 
then,  when  a  few  orange-colored  colonies  may  be  seen, 
these  large  irregular  patches  are  the  most  conspicuous 
objects  on  the  surface  of  the  serum.  Occasionally, 
almost  nothing  else  appears. 

The  cover-slips  made  from  the  membrane  at  the  time 
the  cultures  were  prepared  will  be  found  on  microscopic 
examination  to  present,  in  many  cases,  a  great  variety  of 
organisms ;  but  conspicuous  among  them  will  be  noticed 
slightly  curved  bacilli  of  irregular  size  and  outline. 
In  some  cases  they  will  be  more  or  less  clubbed  at 
one  or  both  ends;  sometimes  they  appear  spindle  in 
shape,  again  as  curved  wedges ;  now  and  then  they  are 
irregularly  segmented.  They  are  rarely  or  never  regu- 
lar in  outline.  If  the  preparation  has  been  stained 
with  Loffler's  alkaline  methylene-blue  solution,  many 
of  these  irregular  rods  are  seen  to  be  marked  by  cir- 
cumscribed points  in  their  protoplasm  which  stain  very 
intensely — they  appear  almost  black.  This  irregularity 
in  outline  is  the  morphological  characteristic  of  bacillus 
diphtherice  of  Loffler. 

It  must  be  remembered,  however,  that  the  diagnosis 
of  diphtheria  should  not  under  all  circumstances  be 
made  from  the  examination  of  cover-slip  preparations 
alone,  especially  when  they  are  stained  only  by  the 


BACILLUS  DIPHTHERIA.  351 

usual  method — i.  e.,  with  Loffler's  methylene-blue. 
There  are  other  organisms  present  in  the  mouth-cavity, 
particularly  in  the  mouths  of  persons  having  decayed 
teeth,  the  morphology  of  which  is  so  like  that  of  the 
bacillus  of  diphtheria  that  they  might  easily  be  mistaken 
tor  that  organism  if  subjected  to  only  the  usual  method 
of  microscopic  examination ;  and  again,  the  genuine  diph- 
theria bacillus  is  sometimes  found  in  the  mouth-cavities 
of  healthy  persons  in  attendance  upon  diphtheria  cases, 
such  persons  being  at  the  time  insusceptible  to  the  patho- 
genic activities  of  the  organism.  In  the  vast  majority 
of  instances,  however,  where  the  clinical  condition  of 
the  patient  justifies  a  suspicion  of  diphtheria,  a  micro- 
scopic examination  alone  of  the  deposit  in  the  throat 
will  serve  to  confirm  or  contradict  this  opinion,  and 
such  examinations  very  frequently  reveal  the  diph- 
theritic nature,  etiologically  speaking,  of  mild  condi- 
tions of  the  throat  which  are  not  associated  with  grave 
constitutional  manifestations. 

Bacillus  diphtherias,  discovered  microscopically  by 
Klebs,  and  isolated  in  pure  culture  and  proved  to 
stand  in  causal  relation  to  diphtheria  by  Loffler,  can 
readily  be  identified  by  its  cultural  peculiarities  and 
by  its  pathogenic  activity  when  introduced  into  tissues 
of  susceptible  animals.  In  guinea-pigs  and  kittens  the 
results  of  its  growth  are  histologically  identical  with 
those  found  in  the  bodies  of  human  beings  who  have 
died  of  diphtheria. 

When  studied  in  pure  culture  its  morphological  and 
cultural  peculiarities  are  as  follows  : 

MORPHOLOGY. — As  obtained  directly  from  the  diph- 
theritic deposit  in  the  throat  of  an  individual  sick  of 
the  disease,  it  is  sometimes  comparatively  regular  in 


352  BACTERIOLOGY. 

shape,  appearing  as  straight  or  slightly  curved  rods  with 
more  or  less  pointed  ends.  More  frequently,  however, 
spindle-  and  club-shapes  occur,  and  not  rarely  many  of 
these  rods  stain  irregularly ;  in  some  of  them  very 
deeply  stained  round  or  oval  points  can  be  detected. 

When  cultures  are  examined  microscopically  it  is 
especially  characteristic  to  find  irregular,  bizarre  forms, 
such  as  rods  with  one  or  both  ends  swollen,  and  very 
frequently  rods  broken  at  irregular  intervals  into  short, 
sharply  defined  segments,  either  round,  oval,  or  with 
straight  sides.  Some  forms  stain  uniformly,  others  in 
various  irregular  ways,  the  most  common  being  the 
appearance  of  deeply  stained  granules  in  a  lightly 
stained  bacillus. 

By  a  series  of  studies  upon  this  organism  when  cul- 
tivated under  artificial  conditions  we  have  found  that 
its  form  and  size  depend  very  largely  upon  the  nature 
of  its  environment.  That  is  to  say,  its  morphology  is 
always  more  regular,  and  it  is  smaller  on  glycerin-agar- 
agar  than  on  other  media  used  for  its  cultivation  ;  while 
upon  Loffler's  blood-serum  the  other  extremes  of  de- 
velopment appear :  here  one  sees,  instead  of  the  very 
short,  spindle,-  lancet,-  club-shaped,  always  segmented 
and  regularly  staining  forms  as  seen  upon  glycerin-agar- 
agar,  long,  sometimes  extremely  slender,  sometimes 
thicker,  irregularly  staining  threads  that  are  usually 
clubbed  and  frequently  pointed  at  their  extremities. 
They  are,  as.  a  rule,  marked  by  areas  that  stain  more 
intensely  than  does  the  rest  of  the  rod,  and  at  times  they 
may  be  a  little  swollen  at  the  centre.  These  differences 
are  so  conspicuous  that  microscopic  preparations  from 
cultures  from  the  same  source,  but  cultivated  in  the  one 
case  on  glycerin-agar-agar  and  in  the  other  upon  blood- 


BACILLUS  DIPHTHERIA.  353 

serum,  when  placed  side  by  side  would  hardly  be  retog- 
ni/ed  as  of  the  same  organism,  unless  its  peculiar  be- 
havior under  these  circumstances  was  already  known. 
Another  peculiar  variation  is  that  observed  upon  very 
slightly  acid  blood-serum.  Here  the  rods  appear 
swollen,  and  are  usually  contracted  to  oval  or  short, 
oblong  bodies,  which  stain  very  faintly,  and  in  which 
are  usually  located  one  or  two  very  deeply  staining 
round  or  oval  points.  Various  authors  'have  called 
attention  to  branching  forms  of  this  organism  that  are 
occasionally  encountered,  especially  when  cultivated 
upon  albumin.  We  have  never  seen  the  branching 
diphtheria  bacilli;  and  in  approximately  9000  blood- 
serum  cultures  from  cases  of  diphtheria  that  have  been 
examined  by  competent  bacteriologists  at  the  laboratory 
of  the  Board  of  Health  of  Philadelphia,  the  branching 
forms  of  this  organism  were  not  observed  in  a  single 
instance.  It  is  fair  to  assume,  therefore,  that  this  pecu- 
liar morphological  variation  of  bacillus  diphtherice  is 
comparatively  rare. 

On  plain  nutrient  agar-agar  (that  is,  nutrient  agar- 
agar  without  glycerin) ;  on  solidified  egg-albumin ;  on 
a  medium  consisting  of  dried  commercial  albumin 
dissolved  in  bouillon  (about  10  grammes  of  albumin 
to  100  c.c.  of  bouillon  containing  1  per  cent,  of  grape- 
sugar)  ;  in  bouillon  without  glycerin,  and  in  bouil- 
lon to  which  a  bit  of  hard-boiled  egg  has  been  added, 
the  morphology  of  the  organism  is  about  intermediate, 
in  both  size  and  outline,  between  the  forms  seen  upon 
glycerin-agar-agar  and  upon  Loffler's  blood-serum. 
There  will  appear  about  an  equal  number  of  short  seg- 
mented and  longer,  irregularly  staining  forms ;  but  in 
general  the  longest  are  rarely  as  long  as  the  long  forms 

23 


354 


BACTERIOLOGY. 


seen  on  blood-serum,  and  throughout  they  are  not  so 
conspicuous  for  the  irregularity  of  their  staining. 

In  cultures  made  upon  two  sets  of  nutrient  agar-agar 
tubes,  differing  only  in  the  fact  that  one  set  contains 
glycerin  to  the  extent  of  6  per  cent.,  while  the  other  set 
contains  none,  a  noticeable  difference  in  morphology  can 


FIG.  66. 


A. 


Bacillus  diphtherix.  A.  Its  morphology  on  glycerin-agar-agar.  B.  Its  mor- 
phology on  Lomer's  blood-serum,  c.  Its  morphology  on  acid  blood-serum 
mixture. 

usually  be  made  out :  while  the  forms  on  the  glycerin- 
agar-agar  cultures  are  throughout  small,  and  pretty 
regular  in  size,  shape,  and  staining,  those  on  the  plain 
agar-agar  are  larger,  stain  less  uniformly,  vary  more  in 
shape,  and  when  stained  by  Loffler's  blue  are  not  so 
regularly  marked  by  pale  transverse  lines  that  give  to 
them  the  appearance  of  being  made  up  of  numerous 
short  segments. 


BACILLUS  DIPHTHERIA.  355 

Though  the  outline  of  this  organism  is  more  regular 
under  some  circumstances  than  others,  it  is  nevertheless 
always  conspicuous  for  its  manifold  variations  in  shape. 

GROWTH  ON  SERUM  MIXTURE. — The  medium  upon 
which  bacillus  diphtheria?  grows  most  rapidly  and  lux- 
uriantly, and  which  is  best  adapted  for  determining  its 
presence  in  diphtheritic  exudates,  is,  as  has  been  stated, 
the  blood-serum  mixture  of  Loffler.  (See  chapter  on 
Media.)  On  the  blood-serum  mixture  the  colonies  of 
baeittus  diphtherice  grow  so  much  more  rapidly  than  the 
other  organisms  usually  present  in  secretions  and  exuda- 
tions in  the  throat  that  at  the  end  of  twenty-four  hours 
they  are  often  the  only  colonies  that  attract  attention  ;  and 
if  others  of  similar  size  are  present,  they  are  generally  of 
quite  a  different  aspect.  Its  colonies  are  large,  round, 
elevated,  grayish-white  or  yellowish,  with  a  centre  more 
opaque  than  the  slightly  irregular  periphery.  The  sur- 
face of  the  colony  is  at  first  moist,  but  after  a  day  or 
two  becomes  rather  dry  in  appearance. 

A  blood-serum  tube  studded  with  coalescent  or 
scattered  colonies  of  this  organism  is  so  characteristic 
that  one  familiar  with  the  appearance  can  anticipate 
with  tolerable  certainty  the  results  of  microscopic  exam- 
ination. 

GLYCERIN- AGAR-AGAR.  —  Upon  nutrient  glycerin- 
agar-agar  the  colonies  likewise  present  an  appearance 
that  may  readily  be  recognized.  They  are  in  every 
way  more  delicate  in  structure  than  when  on  the  serum 
mixture.  They  appear  at  first,  when  on  the  sur- 
face, as  very  flat,  almost  transparent,  dry,  non-glisten- 
ing, round  points  which  are  not  elevated  above  the 
surface  upon  which  they  are  growing.  When  slightly 
magnified  they  are  seen  to  be  granular,  and  to  present 


356  BACTERIOLOGY. 

an  irregular  central  marking,  which  is  denser  and  darker 
by  transmitted  light  than  the  thin,  delicate  zone  which 
surrounds  it.  As  the  colony  increases  in  size  the  thin 
granular  peripheral  zone  becomes  broader,  is  usually 
marked  by  ridges  or  cracks,  and  its  periphery  is  notched 

FIG.  67. 


Colonies  of  bacillus  diphtherix  on  glycerin-agar-agar.  a.  Colonies  located 
in  the  depths  of  the  medium,  b.  Colonies  just  breaking  out  upon  the  surface 
of  the  medium,  c.  Fully  developed  surface-colony. 

or  scalloped.  (Fig.  67,  c.)  These  colonies  are  always 
quite  dry  in  appearance.  When  deep  down  in  the  agar- 
agar  they  are  coarsely  granular.  (Fig.  67,  a.)  They 
rarely  exceed  3  mm.  in  diameter. 

GELATIN. — On  gelatin  the  colonies  develop  much 
more  slowly  than  on  media  that  can  be  retained  at  a 
higher  temperature.  They  rarely  present  their  char- 
acteristic appearances  on  gelatin  in  less  than  seventy- 
two  hours.  They  then  appear  as  flat,  dry,  translucent 
points,  usually  round  in  outline. 

When  magnified  slightly  the  centre  is  seen  to  be  more 
dense  than  the  surrounding  zone  or  zones,  for  they  are 
sometimes  marked  by  a  concentric  arrangement  of 
zones.  The  periphery  is  irregularly  notched.  Like 
the  colonies  seen  on  agar-agar,  they  are  granular,  but 
are  much  more  granular  when  seen  in  the  depths  of  the 
gelatin  than  when  on  its  surface.  On  gelatin  the  col- 


BACILLUS  DTPHTHERIM  357 

onies  rarely  become  very  large ;  usually  they  do  .not 
exceed  1.5  mm.  in  diameter. 

BOUILLON. — In  bouillon  it  usually  grows  in  fine 
clumps,  which  fall  to  the  bottom  of  the  tube,  or  become 
deposited  on  its  sides  without  causing  diffuse  clouding 
of  the  bouillon.  Sometimes  there  are  exceptions  to 
this  naked-eye  appearance :  the  bouillon  may  be  dif- 
fusely clouded ;  but  if  one  inspect  it  very  closely,  par- 
ticularly if  he  examine  it  microscopically  as  a  hanging 
drop,  the  arrangement  in  clumps  will  always  be  de- 
tected, but  the  clumps  are  so  small  as  not  to  be  dis- 
cernible by  the  unaided  eye. 

In  bouillon  kept  at  a  temperature  of  35°-37°  C.  a 
soft,  whitish  pellicle  often  forms  upon  the  surface. 

Changes  in  reactions  of  the  bouillon.  The  reaction  of 
the  bouillon  frequently  becomes  at  first  acid,  and  sub- 
sequently again  alkaline,  changes  which  can  be  observed 
in  cultivations  in  bouillon  to  which  a  little  rosolic  acid 
has  been  added.  This  play  of  reactions  has  been  attrib- 
uted to  the  primary  fermentation  of  the  muscle-sugar 
often  present  in  the  bouillon.  It  does  not  occur  when 
the  medium  is  free  from  carbohydrates. 

POTATO. — On  potato  at  a  temperature  of  35°-37°  C. 
its  growth  after  several  days  is  invisible,  only  a  thin, 
dry  glaze  appearing  at  the  point  at  which  the  potato 
was  inoculated.  Microscopic  examination  of  scrapings 
from  the  potato,  after  twenty-four  hours  at  35°-37°  C., 
reveals  a  decided  increase  in  the  number  of  individual 
organisms  planted. 

STAB-  AND  SLANT-CULTURES. — In  stab-  and  slant- 
cultures  on  both  gelatin  and  glycerin-agar-agar  the  sur- 
face-growth is  seen  to  predominate  over  that  along  the 
track  of  the  needle  in  the  depths  of  the  media. 


358  BACTERIOLOGY. 

Isolated  colonies  on  the  surface  of  either  of  the  media 
in  this  method  of  cultivation  present  the  same  charac- 
teristics that  have  been  given  for  the  colonies  on  plates. 

The  growth  in  simple  stab-cultures  does  not  extend 
laterally  very  far  beyond  the  point  at  which  the  needle 
entered  the  medium. 

It  is  a  non-motile  organism. 

It  does  not  form  spores. 

It  is  killed  in  ten  minutes  by  a  temperature  of  58°  C. 

It  grows  at  temperatures  ranging  from  22°  to  37° 
C.,  but  most  luxuriantly  at  the  latter  temperature. 

Its  growth  in  the  presence  of  oxygen  is  more  active 
than  when  this  gas  is  excluded. 

STAINING. — In  cover-slip  preparations  made  either 
from  the  fauces  of  a  diphtheritic  patient  or  from  a  pure 
culture  of  the  organism  it  is  seen  to  stain  readily  with 
the  ordinary  aniline  dyes.  It  stains  also  by  the  method 
of  Gram,  but  the  best  results  are  obtained  by  the  use 
of  Loffler's  alkaline  methylene-blue  solution  ;  this  brings 
out  the  dark  points  in  the  protoplasmic  body  of  the 
bacilli  and  thus  aids  in  their  identification. 

For  the  purpose  of  demonstrating  the  Loffler  bacil- 
lus in  sections  of  diphtheritic  membrane,  both  the  Gram 
method  and  the  fibrin  method  of  Weigert  give  excellent 
results. 

PATHOGENIC  PROPERTIES. — When  inoculated  sub- 
cutaneously  into  the  bodies  of  susceptible  animals  the 
result  is  not  the  production  of  septicaemia,  as  is  seen  to 
follow  the  introduction  into  animals  of  certain  other 
organisms  with  which  we  shall  have  to  deal,  but  the 
bacillus  of  diphtheria  remains  localized  at  the  point 
of  inoculation,  rarely  disseminating  further  than  the 
nearest  lymphatic  glands.  It  develops  at  the  point  in 


BACILLUS  DIPHTHERIA.  359 

the  tissues  at  which  it  is  deposited,  and  during  its. de- 
velopment gives  rise  to  changes  in  the  tissues  which 
result  entirely  from  the  absorption  of  poisonous  albu- 
mins generated  by  the  bacilli  in  the  course  of  their  de- 
velopment. 

In  a  certain  number  of  cases l  diphtheria  bacillr  have 
been  found  in  the  blood  and  internal  organs  of  individ- 
uals dead  of  the  disease  ;  but  all  that  has  been  "learned 
from  careful  study  of  the  secondary  manifestations  of 
diphtheria  tends  to  the  opinion  that  they  are  in  no  way 
dependent  upon  the  immediate  presence  of  bacteria,  and 
that  the  occasional  appearance  of  diphtheria  bacilli  in 
the  internal  organs  is  in  all  probability  accidental,  and 
usually  unimportant. 

By  special  methods  of  inoculation 2  (the  injection  of 
fluid  cultures  into  the  testicles  of  guinea-pigs)  diph- 
theria bacilli  can  be  caused  to  appear  in  the  omentum ; 
but  this  is  purely  an  artificial  manifestation  of  the  dis- 
ease, and  one  that  is  probably  never  encountered  in  the 
natural  course  of  events.  More  rarely  similar  results 
follow  upon  subcutaneous  inoculation. 

If  a  very  minute  portion  of  either  a  solid  or  fluid 
pure  culture  of  this  organism  be  introduced  into  the 
subcutaneous  tissues  of  a  guinea-pig  or  kitten,  death  of 
the  animal  ensues  in  from  twenty-four  hours  to  five 
days.  The  usual  changes  are  an  extensive  local  oedema, 
with  more  or  less  hypersemia  and  ecchymoses  at  the 

1  Froseh :  "  Die  Verbreitung  des  Diphtheric-bacillus  in  Korper  des 
Menschen,"  Zeit.  fur  Hygiene  und  Infektionskrankheiten,  1893,  Bd. 
xiii.  pp.  49-52.     Booker :  Archives  of  Pediatrics,  Aug.  1893.    Wright 
and  Stokes :  Boston  Med.  and  Snrg.  Journ.,  March  and  April,  1895. 

2  Abbott  and  Ghriskey :  "  A  Contribution  to  the  Pathology  of  Experi- 
mental Diphtheria,"  The  Johns  Hopkins  Hospital  Bulletin,   No.  30, 
April,  1893. 


360  BACTERIOLOGY. 

site  of  inoculation ;  swollen  and  reddened  lymphatic 
glands ;  increased  serous  fluid  in  the  peritoneum,  pleura, 
and  pericardium ;  enlarged  and  hemorrhagic  adrenal 
bodies ;  occasionally  slightly  swollen  spleen  ;  and  some- 
times fatty  degeneration  in  the  liver,  kidney,  and  myo- 
cardium. In  guinea-pigs,  especially,  the  liver  often 
shows  numerous  macroscopic  dots  and  lines  on  the  sur- 
face and  penetrating  the  substance  of  the  organ.  They 
vary  in  size  from  a  pin-point  to  a  pin-head,  and  may  be 
even  larger.  They  are  white  and  do  not  project  above 
the  surface  of  the  capsule. 

The  bacilli  are  always  to  be  found  at  the  site  of  inoc- 
ulation, most  abundant  in  the  grayish-white,  fibrino- 
purulent  exudate.  They  become  fewer  at  a  distance 
from  this,  so  that  the  more  remote  parts  of  the  cedema- 
tous  tissues  do  not  contain  them.  They  are  found  not 
only  free,  but  contained  in  large  number  in  leucocytes, 
some  of  which  have  fragmented  nuclei,  or  have  lost 
their  nuclei.  The  bacilli  within  leucocytes,  as  well  as 
some  outside,  frequently  stain  very  faintly  and  irregu- 
larly, and  may  appear  disintegrated  and  dead. 

Culture-tubes  inoculated  from  the  blood,  spleen,  liver, 
kidneys,  adrenal  bodies,  distant  lymphatic  glands,  and 
serous  transudates,  generally  yield  negative  results  ;  and 
negative  results  are  also  obtained  when  these  organs  are 
examined  microscopically  for  the  bacilli. 

Microscopic  examination  of  the  tissues  at  the  site  of 
inoculation,  as  well  as  of  the  liver,  spleen,  kidneys, 
lymphatic  glands,  and  elsewhere,  reveals  the  presence 
of  localized  foci  of  cell-death,  characterized  by  a  pecu- 
liar fragmentation  of  the  nuclei  of  the  cells  of  these 
parts. 

This  destruction  of  nuclei  results  in  the  formation 


BACILLUS  DIPHTHERIA.  361 

of  groups  of  irregularly  shaped,  deeply  staining  bodies, 
having  at  times  the  appearance  of  particles  of  dust, 
while  again  they  may  be  much  larger.  Some  of  them, 
are  tolerably  regular  in  outline,  while  others  are  irregu- 
larly crescentic,  dumb-bell,  flask-shape,  whetstone- 
shape,  or  bladder-like  in  form.  Occasionally  nuclei 
having  the  appearance  of  being  pinched  or  drawn  out 
can  be  seen.  At  some  points  the  fragments  are  grouped 
in  isolated  masses,  indicating  the  location  of  the 
nucleus  from  the  destruction  of  which  they  originated. 
These  particles  always  stain  much  more  intensely  than 
do  the  normal  nuclei  of  the  part.1  Oertel  has  shown 
that  these  peculiar  alterations  in  their  distribution  are 
characteristic  of  human  diphtheria,  and  the  demon- 
stration of  similar  changes  in  animals  inoculated  with 
this  organism  is  important  additional  proof  that  diph- 
theria is  caused  by  it. 

By  the  inoculation  of  certain  animals  an  affec- 
tion may  be  produced  in  all  respects  identical  with 
diphtheria  as  it  exists  in  man.  If  one  open  the 
trachea  of  a  kitten  and  rub  upon  the  mucous  mem- 
brane a  small  portion  of  a  pure  culture  of  this  organ- 
ism, the  death  of  the  animal  usually  ensues  in  from  two 
to  four  days.  At  autopsy  the  wound  will  be  found 
covered  with  a  grayish,  adherent,  necrotic,  distinctly 
diphtheritic  layer.  Around  the  wound  the  subcuta- 
neous tissues  will  be  redernatous.  The  lymphatic  glands 
at  the  angle  of  the  jaws  will  be  swollen  and  reddened. 
The  mucous  membrane  of  the  trachea  at  the  point  upon 

1  See  "  The  Histological  Changes  in  Experimental  Diphtheria,"  also 
"The  Histological  Lesions  Produced  by  the  Toxalbumin  of  Diph- 
theria," by  Welch  and  Flexner,  Johns  Hopkins  Hospital  Bulletin, 
August,  1891,  and  March,  1892. 


362  BACTERIOLOGY. 

which  the  bacilli  were  deposited  will  be  covered  with 
a  tolerably  firm,  grayish-Avhite,  loosely  attached  pseudo- 
membrane  in  all  respects  identical  with  the  croupous 
membrane  observed  in  the  same  situation  in  cases  of 
human  diphtheria.  In  the  pseudo-membrane  and  in 
the  oadematous  fluid  about  the  skin-wound  bacillus 
diphtherias  may  be  found  both  in  cover-slips  and  in 
cultures. 

From  what  we  have  seen — the  localization  of  the 
bacilli  at  the  point  of  inoculation,  their  absence  from 
the  internal  organs,  and  the'  changes  brought  about  in 
the  cellular  elements  of  the  internal  organs — there  is 
but  one  interpretation  for  this  process,  viz.,  that  it  is 
due  to  the  production  of  a  soluble  poison  by  the  bac- 
teria growing  at  the  site  of  inoculation,  which,  gaining 
access  to  the  circulation,  produces  the  changes  that  we 
observe  in  the  tissues  of  the  internal  viscera. 

This  poison  has  been  isolated  from  cultures  of  ba- 
cillus diphtherice,  and  is  found  to  belong,  not  to  the 
crystallizable  ptomaines,  but  to  the  toxic  albumins — 
bodies  which,  in  their  chemical  composition,  are  analo- 
gous to  the  poison  of  certain  venomous  serpents.  By 
the  introduction  of  this  toxalbumin,  as  it  is  called,  into 
the  tissues  of  guinea-pigs  and  rabbits  the  same  patho- 
logical alterations  may  be  produced  that  we  have  seen 
to  follow  inoculation  with  the  bacilli  themselves,  except, 
perhaps,  the  production  of  false  membranes. 

Under  certain  circumstances  with  which  we  are 
not  acquainted  bacillus  diphtherice  becomes  diminished 
in  virulence  or  may  lose  it  entirely,  so  that  it  is  no 
longer  capable  of  producing  death  of  susceptible  ani- 
mals, and  may  cause  only  a  transient  local  reaction 
from  which  the  animal  entirely  recovers.  Sometimes 


BACILLUS  DIPHTHERIA.  363 

this  reaction  is  so  slight  as  to  be  overlooked,  and 
apiiu  careful  search  may  fail  to  reveal  evidence  of 
any  reaction  at  all.  This  exhibition  of  the  extremes  of 
its  pathogenic  properties,  viz.,  death  of  the  animal,  on 
the  one  hand,  and  only  very  slight  local  effects  on  the 
other,  was  at  one  time  thought  to  indicate  the  existence 
of  two  separate  and  distinct  organisms  that  were  alike 
in  cultural  and  morphological  peculiarities,  but  which 
differed  in  their  disease-producing  power.  Further 
studies  on  this  point  have,  however,  shown  that  the 
genuine  bacillus  diphtherice  may  possess  almost  all 
grades  of  virulence,  and  that  absence  of  or  dimi- 
nution in  virulence  can  hardly  serve  to  distinguish 
as  separate  species  those  varieties  that  are  otherwise 
alike ;  moreover,  the  histological  conditions  found  at  the 
site  of  inoculation  in  animals  that  have  not  succumbed, 
but  in  which  only  the  local  reaction  has  appeared,  are 
in  most  cases  characterized  by  the  same  changes  that 
are  seen  at  autopsy  in  animals  in  which  inoculation  has 
proved  fatal. 

In  the  course  of  their  observations  upon  a  large 
number  of  cases  Roux  and  Yersin  found  that  it  was 
not  difficult  to  detect,  in  the  diphtheritic  deposits  of 
the  same  individual,  bacilli  of  identical  cultural  and 
morphological  peculiarities,  but  of  very  different  degrees 
of  virulence,  and  that  with  the  progress  of  the  disease 
toward  recovery  the  less  virulent  varieties  often  became 
quite  frequent.1 

There  is,  moreover,  a  mild  form  of  diphtheria,  etio- 

1  It  must  not  be  assumed  from  this  that  the  bacilli  lose  their  viru- 
lence entirely,  or  that  they  all  become  attenuated  with  the  establish- 
ment of  convalescence,  for  this  is  contrary  to  what  experience  has 
shown  to  be  the  case. 


364  BACTERIOLOGY. 

logically  speaking,  affecting  only  the  mucous  membrane 
of  the  nares,  known  as  membranous  rhinitis,  from 
which  it  is  very  common  to  obtain  cultures  in  all  re- 
spects identical  with  those  from  typical  diphtheria,  save 
for  their  inability  to  kill  susceptible  animals.  On  inocu- 
lation these  cultures  produce  only  local  reactions,  but 
these  are  characterized  histologically  by  the  same  kind 
of  tissue-changes  that  follow  inoculation  with  the  fully 
virulent  organism. 

Clinically,  membranous  rhinitis  is  never  such  an 
alarming  disease  as  is  laryngeal  or  pharyngeal  diph- 
theria, and,  as  stated,  the  organisms  causing  it  are  often 
of  a  low  degree  of  virulence,  though  they  are,  never- 
theless, genuine  diphtheria  bacilli. 

For  those  organisms  that  are  in  all  respects  identical 
with  the  virulent  bacillus  diphtherice,  save  for  their  ina- 
bility to  kill  guinea-pigs,  the  designation  "  pseudo-diph- 
theritic bacillus "  is  usually  employed ;  but  from  such 
observations  as  those  just  cited  we  are  inclined  to  the 
opinion  that  j^sonio-diphtheritic,  as  applied  to  an  organ- 
ism in  all  respects  identical  with  the  genuine  bacillus, 
except  that  it  is  not  fatal  to  susceptible  animals,  is  a 
misnomer,  and  that  it  would  be  more  nearly  correct  to 
designate  this  organism  as  the  attenuated  or  non- viru- 
lent diphtheritic  bacillus,  reserving  the  term  "pseudo- 
diphtheritic  "  for  that  organism  or  group  of  organisms 
(for  there  are  probably  several)  that  is  enough  like  the 
diphtheria  bacillus  to  attract  attention,  but  is  distin- 
guishable from  it  by  certain  morphological  and  cultural 
peculiarities  aside  from  the  question  of  virulence. 

It  is  a  well-known  fact  that  many  pathogenic  organ- 
isms— conspicuous  among  these  being  micrococcus  lan- 
ceolatus,  staphylococcus  pyogenes  aureus,  streptococcus 


PSEUDO-DIPHTHERITIC  BACILLUS.  365 

pyogenes,  and  the  group  of  so-called  "  hemorrhagic 
septicaemia  "  organisms — undergo  marked  variations  in 
their  pathogenic  properties;  and  yet  these  organisms, 
when  found  either  devoid  of  this  peculiarity,  or  possess- 
ing it  in  a  diminished  degree,  are  not  designated  as 
"pseudo"  forms,  but  simply  as  the  organisms  them- 
selves, the  virulence  of  which,  from  various  causes,  has 
been  modified. 

It  must  nevertheless  be  admitted  that  in  the  course 
of  microscopic  examination  of  materials  from  various 
sources,  including  the  pharynx,  one  occasionally  en- 
counters micro-organisms  whose  morphology  is  so  like 
that  of  the  genuine  bacillus  diphtherice  as  to  create 
suspicion,  and  yet  they  are  at  the  same  time  sufficiently 
unlike  it  to  make  one  cautious  in  forming  an  opinion 
as  to  their  real  nature.  In  the  routine  examination 
of  such  large  numbers  of  cases  as  are  submitted  for 
diagnosis  to  municipal  laboratories  it  is  not  practi- 
cable to  subject  all  specimens  to  the  crucial  animal- 
test,  and  for  this  reason  there  can  be  no  doubt  that  the 
results  of  microscopic  examination  alone  were  formerly 
occasionally  incorrect.  Through  the  suggestion  of 
Neisser1  we  are  fortunately  enabled  to  overcome  this 
difficulty  in  very  large  part.  He  has  found  that  by 
the  use  of  a  particular  staining  method  the  appear- 
ance of  bacillus  diphtheria  is  strikingly  unlike  that  of 
the  confusing  forms.  His  differential  method  compre- 
hends the  following  manipulations :  the  culture  to  be 
tested  should  be  grown  upon  Loffler's  blood-serum 
mixture  solidified  at  100°  C. ;  it  should  develop  at  a 
temperature  not  lower  than  34°  C.  and  not  higher 

1  Neisser :  Zeitschrift  fiir  Hygiene  und  Infektionskrankheiten, 
1897,  Bd.  xxiv. 


366 


BACTERIOLOGY. 


than  36°  C. ;  and  it  should  be  not  younger  than  nine 
and  not  older  than  twenty-four  hours.  A  cover-glass 
preparation  made  from  such  a  culture  is  stained  as 
follows : 

a.  It  is  subjected  to  the  following  mixture  for  from 
one  to  three  seconds : 


Methylene-blue  (Griibler's)  . 
Alcohol  (96  per  cent.)   .    .    . 

When  dissolved,  mix  with 


1  gramme. 

20  c.c. 


Acetic  acid 50  c.c. 

Distilled  water 950  c.c. 

b.  After  thoroughly  rinsing  in  water,  it  is  stained  for 
from  three  to  five  seconds  in  vesuvin  (Bismarck-brown), 

FIG.  68. 


Bacillus  diphtherise,  stained  by  Neisser's  method. 

2  grammes,  dissolved  in  1  litre  of  boiling  distilled  water, 
filtered,  and  allowed  to  cool.    It  is  again  rinsed  in  water 


PSEUDO-DIPHTHERITIC  BACILLUS.  367 

and  examined  as  a  water-mount,  or  it  may  be  dried  and 
mounted  in  balsam. 

When  so  treated  the  diphtheria  bacillus  appears  as 
faintly  stained  brown  rods,  in  which  from  one  to  three 
dark-blue  granules  are  always  to  be  observed.  The 
dark  granules  are  at  one  or  both  poles  of  the  cell,  are 
more  or  less  oval,  and  usually  seem  to  bulge  a  little 
beyond  the  contour  of  the  bacillus  in  which  they  are 
located.  (See  Fig.  68.)  From  Neisser's  observations 
and  those  of  others,1  as  well  as  from  personal  experience, 
it  seems  safe  in  the  vast  majority  of  cases  to  regard  all 
bacilli  that  do  not  stain  in  the  way  described  as  distinct 
from  bacillus  diphtheria;.2 

NOTE.— Prepare  cover-slip  preparations  from  the 
mouth-cavities  of  healthy  individuals  and  from  those 
having  decayed  teeth.  Do  they  correspond  in  any  way 
with  those  made  from  diphtheria  ?  Do  the  same  with 
different  forms  of  sore-throat.  Do  the  peculiarities  of 
any  of  the  organisms  suggest  those  of  the  bacillus  of 
diphtheria?  Wherein  is  the  difference? 

In  cultures  and  cover-slips  made  from  both  diph- 
theritic and  from  innocent  sore-throats  are  any  organ- 
isms almost  constantly  present  ?  Which  are  they,  and 
what  are  their  characteristics  ? 

Which  are  the  predominating  organisms  in  the  an- 
ginas of  scarlet  fever? 

Do  these  organisms  simulate,  in  their  cultural  and 
morphological  peculiarities,  any  of  the  different  species 
with  which  you  have  been  working? 

1  Frankel :  Berliner  klin.  Wochenschrift,  1897,  No.  50. 

2  Bergey :  Publications  of  the  University  of  Pennsylvania,  New 
Series,  No.  4,  1898. 


368  BACTERIOLOGY. 

Do  the  diphtheria  bacilli  disappear  from  the  throat 
with  the  disappearance  of  the  membrane?  How  long 
do  they  persist?  When  obtained  from  the  throats  of 
convalescents  are  they  still  pathogenic  for  guinea-pigs  ? 

Prepare  a  bouillon  culture  of  virulent  bacillus  diph- 
therise ;  after  it  has  been  growing  for  thirty-six  hours 
at  37°— 30°  C.  inoculate  a  guinea-pig  subcutaneously 
with  about  0.1  c.c.  of  it.  If  the  animal  dies,  note  care- 
fully the  findings  at  autopsy,  especially  the  distribution 
of  the  bacilli.  Now  add  to  this  culture  sufficient  pure 
carbolic  acid  or  trikresol  to  kill  all  bacteria  in  it,  and 
inject  under  the  skin  of  another  guinea-pig  varying 
amounts  of  the  culture  so  treated,  beginning  with  0.05 
c.c. ;  determine  the  minimum  fatal  dose,  and  note  in 
which  respects  the  post-mortem  findings  simulate  and 
in  which  they  differ  from  those  of  the  first  animal. 
Should  any  of  the  animals  survive  the  injections  of  the 
disinfected  culture,  note  carefully  their  condition  from 
day  to  day,  particularly  any  fluctuations  in  weight. 
When  they  have  quite  recovered  inoculate  them  writh 
living,  virulent  diphtheria  bacilli.  Do  the  results  cor- 
respond with  those  obtained  with  guinea-pigs  that  have 
never  been  treated  at  all  ?  Explain  the  results. 


CHAPTER  XXI. 

Typhoid  fever — Study  of  the  organism  concerned  in  its  production — 
Bacterium  coli  commune — Its  resemblance  to  the  bacillus  of  typhoid 
fever — Its  morphological,  cultural,  and  pathogenic  properties — Its 
differentiation  from  bacillus  typhosus — Methods  of  isolating  the 
typhoid  bacillus. 

BACILLUS  TYPHOSUS. 

THE  organism  which  was  discovered  in  the  tissues  of 
typhoid  cadavers  microscopically  by  Eberth,  and  sub- 
sequently isolated  in  pure  culture  and  described  by 
Gaffky,  is  now  generally  recognized  as  the  etiological 

FIG.  69.  FIG.  70. 


Bacillus  typhosus,  from  culture  Bacillus  typhosus,  showing  flagella 

twenty-four  hours  old,  on  agar-  stained  by  Loffler's  method, 

agar. 

factor  in  the  production  of  typhoid  fever.     It  may  be 
described  as  follows : 

It  is  a  bacillus  about  three  times  as  long  as  broad, 
with  rounded  ends.  It  may  appear  at  one  time  as  very 
short  ovals,  at  another  time  as  long  threads,  and  both 

24  369 


370  BACTERIOLOGY. 

forms  may  occur  together.  Its  breadth  remains  toler- 
ably constant.  Its  morphology  presents  little  that 
will  aid  in  its  identification.  (See  Fig.  69.)  It  stains  a 
trifle  less  readily  with  the  aniline  dyes  than  do  most  of 
the  other  organisms.  It  is  very  actively  motile,  and 
when  stained  by  the  special  method  of  Lbffler  (see 
this  method  in  chapter  on  Staining)  is  seen  to  possess 
very  delicate  locomotive  organs  in  the  form  of  fine, 
hair-like  flagella,  attached  in  large  numbers  to  all  parts 
of  its  surface.  (See  Fig.  70.)  These  flagella  are  not 
seen  in  unstained  preparations,  nor  are  they  rendered 
visible  by  ordinary  methods  of  staining. 

In  patients  suffering  from  typhoid  fever  the  organ- 
ism has  been  found  during  life  in  the  blood,  urine,  and 
faeces,  and  at  autopsies  in  the  tissues  of  the  spleen,  liver, 
kidneys,  intestinal  lymphatic  glands,  and  intestines. 

GELATIN  PLATES. — Its  growth,  'when  seen  in  the 
depths  of  the  medium,  presents  nothing  characteristic, 
appearing  simply  as  round  or  oval,  finely  granular 
points.  On  the  surface  it  develops  as  very  superficial, 
blue- white  colonies,  with  irregular  borders.  They  are 
a  little  denser  at  the  centre  than  at  the  periphery. 


Colony  of  bacillus  typhosus  on  gelatin. 


When  magnified,  the  colonies  present  wrinkles  or  folds, 
which  give  to  them,  in  miniature,  the  appearance  seen 
in  the  relief  maps  made  to  represent  mountainous  dis- 


BACILLUS  TYPHOSUS.  371 

v 

tricts.  (Fig.  71.)  These  colonies  have  sometimes  the 
appearance  of  flattened  pellicles  of  glass-wool,  and 
usually  a  pearl-like  lustre. 

On  AGAR-AGAR  the  colonies  present  nothing  typical. 

STAB-CULTURES.  —  In  stab-cultures  the  growth  is 
mostly  on  the  surface,  there  being  only  a  very  limited 
development  down  the  track  made  by  the  needle.  The 
surface-growth  has  the  same  appearance  in  general  as 
that  given  for  the  colonies. 

POTATO.  —  The  growth  on  potato  is  usually  described 
as  luxuriant  but  invisible,  making  its  presence  evident 
only  by  the  production  of  a  slight  increase  of  moisture 
at  the  inoculated  point,  and  by  a  limited  resistance 
offered  to  a  needle  when  it  is  scraped  across  the  track 
of  growth.  While  this  is  so  in  many  cases,  yet  it  cannot 
be  considered  as  invariable,  for  at  times  this  organism 
develops  more  or  less  visibly  on  potato. 

POTATO-GELATIN.  —  The  growth  is  similar  to  that 
upon  ordinary  nutrient  gelatin. 

MILK.  —  It  does  not  cause  coagulation  when  grown 
in  sterilized  milk. 

BOUILLON.  —  It  causes  uniform  clouding  of  the  bouil- 
lon and  brings  about  a  slightly  acid  reaction. 

INDOL  FORMATION.  —  It  is  customary  to  regard  this 
organism  as  devoid  of  the  power  of  forming  indol  ; 
in  fact,  this  has  hitherto  been  considered  one  of  its 
important  differential  peculiarities.  By  the  usual  meth- 
od- of  cultivation  and  testing  the  indol  reaction  is  not 
observed  in  cultures  of  the  typhoid  bacillus.  It  has 
ivrciitly  been  shown,  however,  by  Peckham,  that  by 
repented  transplantation,  at  short  intervals,  into  either 
Dunham's  peptone  solution,  or,  preferably,  a  freshly 
prepared  alkali-tryptone  solution,  made  from  trypton- 


372  BACTERIOLOGY. 

ized  beef-muscle,  that  the  indol-producing  function 
may  be  induced  in  the  genuine  typhoid  bacillus  obtained 
directly  from  the  spleens  of  typhoid  cadavers.1 

It  does  not  produce  gaseous  fermentation.  On  lactose- 
litmus-agar-agar  it  grows  as  pale-blue  colonies,  causing 
no  reddening  of  the  surrounding  medium ;  though  if 
glucose  be  substituted  for  lactose,  both  the  colonies  and 
the  surrounding  medium  may  become  red.  In  the  fer- 
mentation-tube, in  glucose  or  lactose  bouillon,  no  evo- 
lution of  gas  as  a  result  of  fermentation  occurs. 

It  does  not  form  spores.  The  irregularities  of  stain- 
ing so  commonly  seen  in  this  organism  have  in  some 
instances  led  to  the  belief  that  the  pale,  unstained  por- 
tions of  the  bacilli  indicate  the  presence  of  spores. 
More  reliable  tests,  however,  have  demonstrated  the 
error  of  this  opinion.  (What  is  the  most  trustworthy 
test  of  spore-formation  ?) 

It  grows  at  any  temperature  between  20°  and  38°  C., 
though  more  favorably  at  the  latter  point.  It  is 
very  sensitive  to  high  temperatures,  being  killed  by 
an  exposure  of  ten  minutes  to  60°  C.,  and  in  a  much 
shorter  time  to  slightly  higher  temperatures. 

It  does  not  liquefy  gelatin. 

It  grows  both  with  and  without  oxygen. 

It  does  not  grow  rapidly. 

Owing  to  a  tendency  to  retraction  of  its  protoplasm 
from  the  cell-envelope  and  the  consequent  production 
of  vacuoles  in  the  bacilli,  the  staining  of  this  organism 
is  frequently  more  or  less  irregular.  At  some  points  in 
a  single  cell  marked  differences  in  the  intensity  of  the 

1  A.  W.  Peckham :  "The  Influence  of  Environment  Upon  the 
Biological  Functions  of  the  Colon  Group  of  Bacilli,"  Journal  of  Experi- 
mental Medicine,  1897,  vol.  ii. 


BACILLUS  TYPHOSUS.  373 

staining  will  be  seen,  and  here  and  there  areas  quite 
free  from  color  can  commonly  be  detected.  These 
colorless  portions  are  often  so  sharply  defined  that  they 

FIG.  72. 


Diagrammatic  representation  of  retraction  of  protoplasm,  with  production 
of  pale  points,  in  bacillus  typhosus. 

look  as  if  they  had  been  punched  out  with  a  sharp 
instrument.  (See  Fig.  72.) 

PRESENCE  IN  TISSUES. — It  is  not  easy  to  demonstrate 
this  organism  in  tissues  unless  it  is  present  in  large  num- 
bers. .The  manipulations  to  which  the  sections  are  sub- 
jected in  being  mounted  often  rob  the  bacilli  of  their 
stain,  and  render  them  invisible,  or  nearly  so.  If, 
however,  sections  be  stained  in  the  carbol-fuchsin  solu- 
tion, either  at  the  ordinary  temperature  of  the  room  or 
at  a  higher  temperature  (40°  to  45°  C.),  then  washed 
in  absolute  alcohol,  and  cleared  in  xylol  and  mounted 
in  balsam,  the  bacilli  (particularly  if  the  tissue  be  the 
liver  and  spleen)  can  readily  be  detected,  massed  to- 
gether in  their  characteristic  clumps.  If  used  in  the 
same  way,  the  alkaline  methylene-blue  solution  gives 
also  very  satisfactory  results. 

In  searching  for  the  typhoid  bacilli  in  tissues  this 
peculiar  deposition  in  clumps  must  always  be  borne  in 
mind,  otherwise  much  labor  will  be  expended  in  vain. 
In  tissues  the  typhoid  bacilli  do  not  lie  scattered  about 
in  the  same  way  as  do  the  organisms  in  tissues  from 
certain  other  conditions — septica3mia,  for  instance  ;  they 


374  BACTERIOLOGY. 

are  not  of  necessity  distributed  along  the  course  of  the 
capillaries,  but  are  localized  in  small  clumps  through 
the  organs,  and  it  is  for  these  clumps,  which  are  easily 
detected  under  a  low-power  objective,  that  one  should 
search.  This  peculiar  clumping  of  the  typhoid  bacilli 
in  the  tissues  cannot  be  satisfactorily  explained.  It 
may  possibly  be  due  to  the  specific  clumping  or  agglu- 
tinating influence  that  typhoid  blood  has  been  shown  to 
have  upon  the  typhoid  bacillus,  a  phenomenon  that  is 
readily  demonstrable  in  the  test-tube  or  under  the 
microscope.  In  other  words,  may  it  not  be  simply  the 
result  of  an  intracapillary  "Widal  reaction"?  (See 
Widal  Reaction.) 

When  the  section  is  prepared  for  examination,  if  it 
be  gone  over  with  a  low-power  objective,  one  will 
notice  at  irregular  intervals  little  masses  that  look  in 
every  respect  like  particles  of  staining-matter  which 
have  been  precipitated  upon  the  section  at  that  point. 
When  these  masses  are  examined  with  a  higher  power 
objective  they  will  be  found  to  consist  of  small  ovals  or 
short  rods  so  closely  packed  that  the  individuals  com- 
posing the  clump  can  often  be  seen  only  at  the  extreme 
periphery  of  the  mass.  This  is  the  characteristic  ap- 
pearance of  the  typhoid  organism  in  tissues.  The  little 
masses  are  usually  in  the  neighborhood  of  a  capillary. 

RESULT  OF  INOCULATION  INTO  LOWER  ANIMALS. — 
A  great  many  experiments  have  been  made  in  a  variety 
of  ways  with  the  view  of  reproducing  the  pathological 
conditions  of  this  disease,  as  seen  in  man,  in  the  tis- 
sues of  lower  animals,  but  with  practically  no  success. 
From  the  time  of  its  discovery  up  to  within  a  compara- 
tively recent  date  there  was  an  almost  continuous  con- 
troversy concerning  the  infective  properties  of  bacillus 


BACILLUS  TYPHOSUS.  375 

fi/phosus  for  animals.  By  some  it  was  held  that  the 
effects  of  its  introduction  into  animals  were  manifestly 
of  toxic  l  origin,  while  others  regarded  them  as  evidences 
of  genuine  infection.2  These  diversities  of  opinion  are 
hardly  surprising  when  we  remember  that  animals 
never  suffer  naturally  from  a  disease  similar  to  typhoid 
fever,  and  therefore  offer  many  obstacles  to  its  faithful 
reproduction,  and  that  the  vigor  of  this  organism  when 
cultivated  from  various  sources  is  liable  to  a  wide  range 
of  fluctuation.  For  a  time  there  seemed  to  be  good 
grounds  for  the  opinion  that  under  exceptional  circum- 
stances bacillus  typhosus  did  exhibit  truly  infective 
properties,  and  the  reported  experiments  of  Cygnaus3 
in  particular,  as  well  as  a  single  observation  by  the 
writer,4  in  no  wise  weakened  this  opinion.  By  a  variety 
of  methods  Cygnseus  demonstrated  that  this  organism 
possessed  the  property  of  multiplying  within  the  in- 
ternal organs  of  animals  and  of  causing  constitutional 
symptoms  and  pathological  lesions  that  very  closely 
simulated  those  of  typhoid  fever  as  seen  in  man.  In 
1890  the  writer  called  attention  to  the  lesions  found  in 
one  of  a  number  of  rabbits  that  had  succumbed  to 
intravenous  injection  of  large  amounts  of  fluid  cultures 
of  this  organism. 

In  this  case  there  was  an  ulcer  in  the  ileum  which 
was  macroscopically  and  microscopically  identical  with 
found  at  autopsy  in  the  small  intestine  of  human 


1  Toxic—  poisonous  results  not  necessarily  accompanied  by  the  growth 
of  organisms  throughout  the  tissues. 

2  Infective  or  septic—  poisoning  of  the  tissues  as  a  result  of  the  growth 
of  bacteria  within  them. 

'Cygnseus:  Ziegler's  Beitrage  zur  Anat.  und   Path.,  1890,  Bd.  vii. 
Heft  3,  S.  377. 
4  Bulletin  of  the  Johns  Hopkins  Hospital,  1890,  vol.  i.  p.  63. 


376  BACTERIOLOGY. 

subjects  dead  of  this  disease.  The  typhoid  bacilli  were 
not  only  obtained  from  the  spleen  of  the  animal  by 
culture  method,  but  the  characteristic  clumps  were  also 
demonstrated  microscopically  in  sections  of  the  organ. 

It  must  be  said,  however,  that  such  results  are  ex- 
tremely rare.  As  a  rule,  the  only  effects  that  follow 
the  introduction  of  this  organism  into  animals  are  refer- 
able to  the  intoxicating  action  of  the  materials  used. 
In  fact,  the  results  of  modern  investigations  have  placed 
bacillus  typhosus  in  the  category  of  toxin-producers, 
and  through  the  use  of  the  toxins  produced  by  it  ani- 
mals have  been  rendered  immune  from  otherwise  fatal 
doses.  The  serum  of  such  animals  has  also  been  shown 
to  possess  a  certain  degree  of  immunizing  power.1 

In  connection  with  the  inoculation  of  animals  with 
bacillus  typhosus  observations  of  a  most  important 
nature  have  been  made  by  Sanarelli 2  upon  the  arti- 
ficial induction  of  susceptibility  to  its  pathogenic  ac- 
tion. He  found  that  rabbits,  guinea-pigs,  and  mice 
could  be  rendered  susceptible  to  infection  by  this  organ- 
ism by  preliminary  injections  into  them  of  the  products 
of  growth  of  certain  saprophytes — proleus  vulgaris, 
bacillus  prodigiosus,  and  bacterium  coli  commune;  and 
that  by  whatever  means  the  animal  was  subsequently 
inoculated  with  fresh  cultures  of  the  typhoid  bacillus, 
either  into  the  circulation  or  into  the  peritoneal  cavity, 
death  resulted  in  from  twelve  to  forty-eight  hours,  with 
the  pathological  alterations  most  conspicuous  in  the 
digestive  tract,  and  particularly  in  the  small  intestine. 
In  these  cases  the  infection  is  general,  and  the  organisms 

1  Pfeiffer  and  Kolle :  Zeitschrift  fur  Hygiene  und  Infektionskrank- 
heiten,  1896,  Bd.  xxi.  S.  208. 

2  Sanarelli:  Annales  de  1'Institut  Pasteur,  1892,  tome  vi. 


BACILLUS  TYPHOSUS.  377 

may  be  recovered  from  the  blood  and  internal  organs. 
It  is  the  opinion  of  Sauarelli  that  the  toxic  conditions 
produced  by  the  preliminary  injections  of  the  products 
of  growth  of  the  saprophytic  organisms  may  be  consid- 
ered analogous  to  a  similar  condition  that  may  occur 
in  man  from  the  absorption  of  abnormal  products  of 
fermentation  from  the  intestinal  canal — an  auto-intoxi- 
cation that  so  reduces  the  resistance  of  the  individual 
as  to  render  him  susceptible  to  infection  by  the  bacillus 
of  typhoid  fever,  should  it  gain  access  to  his  alimentary 
tract. 

More  recently  it  was  reported  by  Alessi l  that  rats, 
guinea-pigs,  and  rabbits,  when  compelled  to  breathe  the 
gaseous  products  of  decomposition  from  the  contents  of 
a  cesspool,  or  from  other  decomposing  matters,  gradu- 
ally became  susceptible  to  infection  by  the  typhoid 
bacillus;  but  unfortunately  for  the  value  of  this  ob- 
servation the  description  given  by  Alessi  of  the  two 
cultures  of  so-called  typhoid  bacilli  used  by  him  for 
inoculation  was  in  one  case  certainly  not  that  of  the 
typhoid  organism,  and  in  the  other  the  culture  used  had 
been  kept  under  artificial  conditions  so  long  as  hardly 
to  be  reliable  for  tests  of  this  character. 

The  importance  of  these  observations  in  their  bearing 
upon  the  etiology  of  typhoid  fever,  if  they  are  demon- 
strated by  subsequent  experiment  to  be  trustworthy,  is 
too  obvious  to  necessitate  emphasis,  and  it  is  greatly  to 
be  desired  that  they  may  not  be  permitted  to  pass  un- 
noticed, but  that  others  interested  may  find  occasion  to 
institute  experiments  in  the  same  direction,  with  the 
hope  that  some  light  may  be  shed  upon  the  mooted 

1  Alessi :  Centralblatt  fur  Bakteriologie  un.  Parasitenkunde  1894, 
Bd.  xv.  No.  7,  p.  228. 


378  BACTERIOLOGY. 

question  concerning  the  influence  of  gaseous  products 
of  decomposition  upon  the  health  of  individuals,  and 
particularly  upon  the  part  played  by  them  in  diminish- 
ing natural  resistance  to  infection.1 

Because  of  the  variations  in  the  morphology  and  cult- 
ural peculiarities  of  this  organism,  and  because  of  the 
difficulty  experienced  in  efforts  to  reproduce  in  lower 
animals  the  conditions  found  in  the  human  subject, 
typhoid  fever  is  bacteriologically  one  of  the  most  unsat- 
isfactory of  the  infectious  diseases. 

A  number  of  other  organisms  appear  botanically  to 
be  closely  related  to  the  typhoid  bacillus,  and  with  our 
present  methods  for  studying  them  they  so  closely  simu- 
late it  that  the  difficulty  of  identifying  this  organism 
is  sometimes  very  great.  In  addition  the  variability 
constantly  seen  in  pure  cultures  of  the  typhoid  bacillus 
itself  in  no  way  renders  the  task  more  simple. 

For  example,  the  morphology  of  the  typhoid  ba- 
cillus is  conspicuously  inconstant ;  its  growth  on  potato, 
which  was  formerly  described  as  characteristic,  may, 
with  the  same  stock,  at  one  time  be  the  typical  invis- 
ible development,  at  another  time  it  may  grow  in  a  way 
easily  to  be  seen  with  the  naked  eye  ;  and  the  change  of 
reaction  which  it  is  said  to  produce  in  bouillon  is  some- 
times much  more  intense  than  at  others.  The  indol- 
producing  function,  hitherto  regarded  as  absent  from 
this  organism,  is  now  known  to  be  occasionally  de- 
monstrable by  ordinary  methods,  and  frequently  by 
special  methods  of  cultivation.  (Peckham,  I.  c.)  The 

1  See  paper  by  the  author :  "  The  Effects  of  the  Gaseous  Products  of 
Decomposition  upon  the  Health,  and  Resistance  to  Infection,  of  Certain 
Animals  that  are  Forced  to  Respire  Them,"  Transactions  of  the  Asso- 
ciation of  American  Physicians,  1895,  vol.  x.  pp.  16-44. 


BACILLUS  TYPHOSUS.  379 

only  properties  possessed  by  it  that  may  be  said  to 
be  constant  are  its  motility ;  its  inability  to  cause  gas- 
eous fermentation  of  glucose,  lactose,  or  saccharose  ;  its 
incapacity  for  coagulating  milk ;  and  its  growth  on 
gelatin  plates  ;  but  there  are  other  bacilli  which  possess 
these  same  characteristics  to  a  degree  that  renders  their 
differentiation  from  the  typhoid  organism  often  a  matter 
that  requires  the  careful  application  of  all  the  different 
tests. 

An  interesting  reaction  of  the  typhoid  bacillus  is 
seen  when  it  is  brought  in  contact  with  the  blood- 
serum  from  human  beings  sick  of  typhoid  fever,  or  from 
animals  that  have  survived  inoculation  with  cultures  of 
this  organism.  This  reaction  consists  of  a  peculiar 
alteration  in  the  relation  of  the  organisms  to  one 
another  in  the  fluid.  As  ordinarily  seen  in  a  hanging 
drop  of  bouillon,  the  typhoid  bacilli  appear  as  single, 
actively  motile  cells ;  when  to  such  a  drop  a  drop  of 
dilute  serum  from  a  case  of  typhoid  fever  is  added  the 
motility  of  the  organism  gradually  becomes  lessened, 
and  finally  ceases,  and  the  bacteria  congregate  in 
larger  and  smaller  clumps.  The  reaction  may  also  be 
produced  in  another  way,  viz.,  by  adding  to  about  4  or 
5  c.c.  of  a  twenty-four-hour-old  bouillon  culture  of 
typhoid  bacilli  in  a  narrow  test-tube  about  eight  drops 
of  serum  from  a  case  of  typhoid  fever,  after  which  the 
tube  is  placed  in  the  incubator.  After  a  few  hours  the 
normally  clouded  culture  is  seen  to  have  undergone  a 
change;  instead  of  the  diffuse  cloud  caused  by  the 
growth,  the  fluid  is  found  clear  and  contains  within  it 
flocculent  masses  of  the  bacteria  that  have  agglutinated 
together  as  a  result  of  the  specific  action  of  the  serum 
used. 


380  BACTERIOLOGY. 

For  the  hanging-drop  test  sufficient  serum  may  be 
obtained  from  a  needle-prick  in  the  finger,  while  for 
the  test-tube  reaction  a  larger  amount  is  needed ;  this 
may  be  obtained  from  blood  drawn  from  a  superficial 
vein  by  means  of  a  hypodermic  syringe,  or  from  the 
cleansed  skin  by  a  wet-cup,  or,  better  still,  from  a 
small  cantharides  blister. 

It  is  proper  to  state,  however,  that  occasionally  cult- 
ures of  genuine  typhoid  bacilli  are  encountered  that  do 
not  respond  to  this  peculiar  influence  of  typhoid  blood, 
even  though  the  blood  be  tested  at  different  stages  of 
the  disease,  and  even  though  it  causes  the  characteristic 
cessation  of  motion  and  clumping  with  other  cultures  of 
this  organism  upon  which  it  may  be  tried. 

When  employed  conversely — i.  e.,  for  deciding  if 
the  serum  used  is  from  a  case  of  typhoid  fever  or  not — 
the  reaction  constitutes  "WidaPs  serum  diagnosis  of 
typhoid  fever."  For  this  purpose  it  is  often  necessary 
to  test  several  cultures  of  genuine  typhoid  bacilli,  from 
different  sources  and  of  varying  degrees  of  vitality,  be- 
fore a  culture  is  procured  that  gives  the  reaction  most 
conspicuously  and  quickly  with  genuine  typhoid  serum. 
This  culture  is  then  to  be  set  aside,  to  be  used  for  this 
test  with  serums  from  doubtful  cases  of  the  disease. 

WIDAI/S  REACTION  WITH  DRIED  BLOOD. — For  clin- 
ical purposes  it  is  of  importance  to  know  that  this  reac- 
tion can  be  obtained  from  dried  blood — i.  e.,  by  the 
method  suggested  by  Wyatt  Johnston,  of  Montreal. 
In  this  method  a  drop  of  the  blood  to  be  tested,  ob- 
tained by  a  needle-prick  in  the  cleansed  finger  or  lobe 
of  the  ear,  is  collected  on  a  bit  of  clean,  unglazed 
paper  and  allowed  to  dry.  The  paper  is  then  folded, 
kept  free  from  contamination,  and  taken  to  the  labora- 


BACILLUS  TYPHOSUS.  381 

tory.  With  a  medium-size  platinum-wire  loop  a  drop 
of  sterile  bouillon,  water,  or  physiological  salt-solution 
is  gently  rubbed  upon  the  drop  of  dried  blood  until  the 
contents  of  the  loop  are  of  a  dark  amber  color;  this  is 
then  mixed  with  a  drop  of  a  bouillon  culture  of  typhoid 
bacilli  on  a  cover-glass,  which  is  mounted  upon  the 
hollow-ground  slide  as  a  hanging  drop,  when  the  effect 
of  the  diluted  blood  upon  the  culture  can  be  observed 
with  the  microscope.  The  reaction,  if  positive,  should 
occur  within  a  half  hour.  Many  object  to  this  method 
because  it  is  impossible  accurately  to  dilute  the  blood 
by  the  plan  used.  A  number  of  tests  have  shown  us 
that  preparations  made  in  this  way  correspond  roughly 
with  a  fresh-blood  dilution  of  from  1  : 15  to  1  : 20,  as 
determined  by  the  hsemoglobinometer.  In  a  small 
number  of  cases  in  which  parallel  tests  were  made 
with  this  and  with  fresh  fluid  serum  the  results  were 
concordant.  We  are  inclined  to  the  opinion,  however, 
that  in  doubtful  cases,  in  which  all  the  available  clin- 
ical evidence  is  opposed  to  either  the  positive  or  nega- 
tive results  of  the  test,  the  difficulty  is  much  more 
certainly  cleared  away  by  the  use  of  highly  diluted 
and  exactly  diluted  fresh  serum  than  by  this  method. 
Competent  observers  are  of  the  opinion  that  in  all 
such  cases  the  quantity  of  serum  in  the  hanging  drop 
should  be  decreased  until  it  is  present  in  the  proportion 
of  from  (not  less  than)  1  :  50  to  1  :  60,  and  that,  if  after 
exposure  to  this  dilution  for  two  hours  the  bacilli  are 
still  motile  and  not  clumped  together,  or  the  reaction  is 
deficient  in  only  one  or  the  other  of  these  peculiarities, 
the  case  from  which  the  serum  was  obtained  may  be 
safely  regarded  as  not  typhoid  fever. 

Experience  with  the  dry-blood  method  at  the  Mu- 


382  BACTERIOLOGY. 

nicipal  Laboratory  of  Philadelphia  in  more  than 
12,000  examinations  from  about  10,000  febrile  con- 
ditions, leads  us  to  regard  the  culture  used  as  one  of 
the  most  important  factors  in  the  test.  After  deciding 
upon  the  most  suitable  culture  for  the  reaction — and  it 
is  often  necessary  to  try  a  great  number  from  various 
sources — we  have  adopted  the  plan  of  daily  trans- 
planting the  culture  into  fresh  bouillon  and  keep- 
ing it  at  a  temperature  rarely  above  20°-22°  C.  The 
bacilli  grown  under  these  circumstances  are  usually 
somewhat  longer  than  when  cultivated  at  higher  tem- 
perature, and  they  exhibit  a  regular,  gliding  motility 
that  renders  it  more  easy  to  follow  the  individual  cells 
under  the  microscope  than  when  they  possess  the  usual 
active,  darting  motion. 

In  the  group  of  cases  examined  by  us  by  the  dry- 
blood  method,  including  typhoid  and  other  febrile  con- 
ditions, there  is  a  discrepancy  between  the  clinical  and 
the  laboratory  diagnosis  in  from  2  to  3  per  cent,  of  the 
cases  examined. 

In  the  hands  of  all  who  have  carefully  employed 
the  Widal  reaction  for  the  diagnosis  of  typhoid  fever 
the  results  are  reported  to  have  been  almost  uniformly 
satisfactory.  In  the  great  majority  of  cases  the  reac- 
tion is,  so  far  as  experience  indicates,  specific — i.  e.,  a 
typical  reaction  does  not  occur  between  typhoid  serum 
and  organisms  other  than  the  typhoid  bacillus,  nor  be- 
tween the  typhoid  bacillus  and  serums  other  than  those 
of  typhoid  fever.  There  are,  however,  confusing  reac- 
tions— so-called  pseudo-reactions — in  which  more  or 
less  clumping  of  the  bacilli  and  a  diminution  of  motion, 
without  complete  cessation,  are  observed.  These  reac- 
tions have  been  seen  to  occur  with  normal  blood  and 


ISOLATING   THE  TYPHOID  BACILLUS.         383 

with  blood  from  other  febrile  conditions.  It  is  said  by 
Johnston  and  McTaggart1  that  they  can  be  prevented 
if  cultures  of  just  the  proper  degree  of  vitality  are  em- 
ployed ;  and  this  corresponds  with  the  results  of  a  fairly 
wide  personal  experience  with  the  test. 

The  blood  of  certain  animals,  as  well  as  a  number  of 
chemical  substances,  such  as  corrosive  sublimate,  alco- 
hol, salicylic  acid,  resorcin,  and  safranin  in  high  dilution, 
cause  agglutination  of  the  typhoid  bacilli ;  but  the  reac- 
tion is  not  specific,  for  in  most  cases  they  have  the  same 
effect  on  other  motile  bacilli. 

The  method  is  yet  in  the  experimental  stage,  and 
there  are  still  numerous  features  not  entirely  clear. 
In  the  light  of  present  experience,  however,  it  is  fair 
presumptive  evidence  that  the  serum  is  from  a  case 
of  typhoid  fever  when  unmistakable  agglutination  and 
cessation  of  motion  are  seen  in  from  fifteen  to  twenty 
minutes  after  typhoid  bacilli  are  mixed  with  the  serum 
of  a  suspicious  febrile  condition. 

All  the  preceding  points  should  be  borne  in  mind  in 
the  examination  of  drinking-water  supposed  to  be  con- 
taminated by  typhoid  dejections,  for  the  organisms 
which  most  clpsely  approach  the  typhoid  bacillus  in 
growth  and  morphology  are  just  those  organisms  which 
would  appear  in  water  contaminated  from  cesspools — 
/.  e.9  the  organisms  constantly  found  in  the  normal  intes- 
tinal tract.  Even  in  the  stools  of  typhoid-fever  patients 
the  presence  of  these  normal  inhabitants  of  the  intes- 
tinal tract  renders  the  isolation  of  the  typhoid  organisms 
somewhat  troublesome. 

METHODS  OF  ISOLATING  THE  TYPHOID  BACILLUS. 
— Bacillus  typhosus  is  so  variable  in  many  of  its  bio- 

1  Johnston  and  McTaggart :  Montreal  Medical  Journal,  March,  1897. 


384  BACTERIOLOGY. 

logical  peculiarities,  and  is  so  closely  simulated  in  cer- 
tain respects  by  a  group  of  other  organisms  to  which  it 
appears  to  be  botanically  related,  that  its  identification, 
especially  outside  the  infected  body,  is  usually  a  matter 
of  considerable  difficulty  and  uncertainty.  For  these 
reasons  many  efforts  have  been  made  to  discover  specific 
reactions  for  the  organism,  and  with  this  end  in  view 
many  methods  have  been  devised  for  its  isolation  from 
water,  faeces,  sewage,  and  other  matters  believed  to  con- 
tain it.  None  of  them,  however,  has  given  general 
satisfaction,  and  many  have  proved  wholly  untrust- 
worthy. Those  worthy  of  some  degree  of  confidence 
are  as  follows : 

Eisner's  method.  The  medium  employed  is  an  acid 
mixture  of  gelatin,  potato-juice,  and  potassium  iodide. 
It  contains  neither  peptone  nor  sodium  chloride.  On 
this  medium  it  is  claimed  that  the  ordinary,  rapidly 
growing,  liquefying  saprophytes  do  not  develop,  and 
that  the  colon  bacillus  and  typhoid  bacillus  find  it 
favorable  for  growth.  These  are  differentiated  from 
one  another  by  the  macroscopic  and  microscopic  char- 
acter of  their  colonies — i.  e.,  the  growth  of  the  colon 
colonies  differs  little  or  not  at  all  from  that  seen  on 
ordinary  nutrient  gelatin,  while  that  of  the  typhoid 
colonies  is  so  slow  that  they  are  scarcely  visible  at  the 
end  of  twenty-four  hours.  After  forty-eight  hours  the 
latter  appear  under  the  low  power  of  the  microscope  as 
small,  pale,  finely  granular,  almost  transparent  bodies 
easily  distinguished  from  the  coarser,  brownish  colonies 
of  the  colon  bacillus. 

While  the  method  is  useful,  it  has  its  limitations, 
and  is  not  always  reliable.  At  times  colon  colonies 
develop  in  a  manner  that  may  readily  cause  one  to 


ISOLATING   THE  TYPHOID  BACILLUS.         385 

mistake  them  for  typhoid  colonies ;  while  typhoid  col- 
onies may  present  the  characteristics  of  those  due  to  the 
growth  of  the  colon  bacillus.  This  is  especially  the 
case  in  plates  more  than  forty-eight  hours  old  that  have 
been  kept  at  ordinary  room- temperature. 

In  our  experience  the  most  serviceable  feature  of  this 
method  is  the  elimination  of  many  of  the  common  sap- 
rophytes usually  present  in  mixtures  containing  typhoid 
and  colon  bacilli.  The  majority  of  them  do  not  grow 
upon  gelatin  made  by  this  method,  which  will  now  be 
described. 

The  description  given  by  Eisner1  of  the  mode  of 
preparation  of  the  medium  is  so  incomplete  and  unsat- 
isfactory in  most  of  the  important  details  that  prac- 
tically all  who  use  the  method  have  been  obliged 
to  develop  their  own  technique  from  the  general  sug- 
gestions made  in  his  original  communication.  The 
"Eisner  medium"  that  has  given  satisfaction  in  our 
hands  is  prepared  as  follows  :  grate  one  kilogramme  of 
peeled  potatoes  and  allow  to  stand  over  night  in  a 
refrigerator.  Then  press  out  all  juice,  using  an  ordi- 
nary meat-press  for  the  purpose.  Filter  this  fresh 
juice  cold,  to  remove  as  much  of  the  starch-gran- 
ules as  possible ;  if  this  is  not  done,  the  starch  when 
heated  swells  to  such  an  extent  as  to  render  filtration 
almost  impracticable.  Boil  the  filtrate  and  filter  again. 
Test  the  filtrate  for  acidity  by  titrating  10  c.c.  with  a 
decinormal  solution  of  sodium  hydroxide,  the  indicator 
used  being  6  drops  of  the  ordinary  ^  per  cent,  solution 
of  phenolphtalein  in  50  per  cent,  alcohol.  The  acidity 
of  the  juice  should  be  such  as  to  require  3  c.c.  of  a  deci- 

1  Eisner :  Zeitschrift  fur  Hygiene  und  Infektionskrankheiten,  1896, 
Bd.  xxi.  S.  25. 
25 


386  BACTERIOLOGY. 

normal  sodium  hydroxide  solution  to  neutralize  10  c.c.  of 
the  juice.  (Potter.)  If  the  acidity  is  found  to  be  greater 
than  this,  which  is  usually  the  case,  dilute  with  water 
until  the  proper  degree  is  reached.  If  less  than  this, 
the  juice  may  be  concentrated  by  evaporation.  It  is 
desirable  that  this  acidity  should  be  due  to  the  acids  nor- 
mally present  in  the  potato,  and  that  it  should  not  be  arti- 
ficially obtained  by  the  addition  of  other  acids.  Now 
add  10  per  cent,  of  gelatin  (no  peptone  and  no  sodium 
chloride),  dissolve  by  boiling,  and  again  test  the  acidity, 
using  10  c.c.  of  the  mixture  and  phenol  phtalein  as 
before.  Deduct  3  c.c.  (the  acidity  of  the  potato-juice, 
that  is  to  be  maintained)  from  the  number  of  c.c.  of  the 
decinormal  sodium  hydroxide  solution  required  to  neu- 
tralize the  10  c.c.  of  the  gelatin  mixture,  and  from  the 
resulting  figure  calculate*  the  amount  of  normal  solution 
of  sodium  hydroxide  needed  for  the  entire  volume,  and 
add  it.  Boil,  clarify  with  an  egg,  and  filter  through 
paper  in  the  usual  manner.  To  the  filtrate  add  potas- 
sium iodide  in  the  proportion  of  1  per  cent.  Decant 
into  tubes  and  sterilize. 

Stoddart's  method.  The  principle  upon  which  this 
method  is  based  may  be  briefly  stated  as  follows  :  if  the 
typhoid  bacillus  be  cultivated  in  a  semi-solid  nutrient 
medium  at  about  35°  C.,  its  growth  is  not  circumscribed 
or  confined  to  the  immediate  neighborhood  of  the  point 
of  inoculation  ;  whereas  if  a  less  actively  motile  organ- 
ism— the  colon  bacillus,  for  example — be  cultivated 
under  similar  circumstances  there  is  no  apparent  ten- 
dency for  it  to  diffuse  itself  through  the  medium  ;  its  de- 
velopment is  confined  to  the  point  of  inoculation.  In  the 
former  case  the  medium  becomes  clouded  throughout ; 
in  the  latter  it  remains  clear,  except  at  the  immediate, 


ISOLATING  THE  TYPHOID  BACILLUS.         387 

sharply  defined  seat  of  growth.  If  a  mixture  of  the 
two  organisms  be  taken  upon  the  point  of  a  needle  and 
deposited  at  a  single  spot  upon  a  plate  made  from  a 
semi-solid  nutrient  medium,  or  be  planted  as  a  stab- 
culture  in  a  test-tube  of  the  same  medium,  the  growth 
of  the  colon  bacillus  remains  about  the  part  touched,  or 
along  the  track  of  the  needle,  while  that  of  the  typhoid 
bacillus  spreads  as  a  delicate  cloud  for  some  distance 
beyond.  The  medium  recommended  by  Stoddart  for 
the  test  is  a  mixture  of  equal  parts  of  ordinary  meat- 
infusion — peptone-agar-agar  (1  per  cent,  agar-agar)  and 
meat-infusion-peptone-gelatin  (10  per  cent,  gelatin). 
In  this  mixture  the  proportion  of  agar-agar  present  is, 
obviously,  0.5  per  cent.,  and  that  of  the  gelatin  5  per 
cent.  These  amounts  are  sufficient  to  keep  it  just  about 
solid  when  exposed  to  35°  C. 

A  limited  experience  with  the  method  leads  us  to 
regard  it  as  a  useful  addition  to  our  means  for  the  iso- 
lation and  identification  of  the  typhoid  and  typhoid-like 
bacilli.  It  is  not  infallible  in  its  indications. 

It  is  employed  in  two  ways — for  plates  and  for  tube 
cultures,  though  the  former  is  perhaps  the  more  useful. 
Here  the  liquefied  culture  mixture  is  poured  into  a  ster- 
ile Petri  dish  and  allowed  to  gelatinize ;  after  which  it 
is  inoculated  at  a  single  point,  or  at  several  points,  with 
the  pure  culture,  or  with  the  mixture  of  organisms  to 
be  tested,  and  placed  at  35°  C.  The  characteristics  of 
the  growths  of  the  typhoid  and  of  the  colon  bacillus 
are  given  above.1 

Hi&Js  method.  A  short  time  subsequent  to  Stoddart's 
publication  Hiss  called  attention  to  a  similar  application 

1  For  further  details,  see  Stoddart :  Journal  of  Pathology  and  Bac- 
teriology, 1897,  vol.  iv.  p.  429. 


388  BACTERIOLOGY. 

of  a  technical  procedure,  which  in  principle,  with  some 
important  additions,  is  almost  identical  with  that  of 
Stoddart.  In  this  method  also  advantage  is  taken  of 
the  fact  that  in  semi-solid  nutrient  media  the  greater 
motility  of  the  typhoid  bacillus  enables  it  to  diffuse  more 
readily  through  the  medium  than  can  the  less  active 
colon  bacillus.  The  endeavor  of  Hiss  was  to  discover 
a  method  wrhereby  this  peculiarity  would  be  favored, 
or  at  least  not  checked,  in  the  typhoid,  and  more  or 
less  suppressed  in  the  colon  bacillus.  A  series  of  experi- 
ments demonstrated  that  if  peptone  be  omitted  and  glu- 
cose be  added  to  the  semi-solid  medium,  the  absence  of 
the  former  important  nutritive  substance  and  the  excess 
of  acidity  resulting  from  the  fermentation  of  the  latter 
had  only  slight  effect  upon  the  characteristic  develop- 
ment of  the  typhoid  bacillus  (a  diffuse  clouding  of  the 
medium),  while  the  influence  upon  the  growth  of  the 
colon  bacilli  was  to  prevent,  in  many  cases,  their  ten- 
dency to  cloud  the  medium  by  sharply  restricting  their 
growth  to  the  point  at  which  they  were  deposited.1 
The  composition  of  the  medium  used  is : 

Agar-agar 5  grammes. 

Gelatin 80        " 

Liebig's  beef-extract 5 

Sodium  chloride 5 

Glucose     5-10        " 

Water 1000  c.c. 

The  gelatin  should  be  added  after  the  agar-agar  and 
other  ingredients  are  dissolved  ;  the  volume  of  the  mass 
is  then  brought  to  1000  c.c.,  and  finally  the  reaction  is 
corrected.  This  should  be  equivalent  to  a  degree  of 
acidity  that  would  require  15  c.c.  of  a  normal  sodium 

1  Hiss :  Journal  of  Experimental  Medicine,  1897,  vol.  ii.  No.  6,  p.  677. 


ISOLATING   THE  TYPHOID  BACILLUS.         389 

hydroxide  solution  to  the  litre  to  bring  it  to  the  phenol- 
phtalein  neutral  point. 

When  planted  as  stab-cultures  in  this  medium  and 
kept  at  body-temperature  the  growth  of  bacillus  typho- 
sus  appears  simply  as  a  diffuse  cloud,  with  little  or  no 
tendency  to  appear  more  concentrated  along  the  track  of 
the  needle ;  while  the  development  of  the  colon  bacillus 
is  confined  to  the  neighborhood  of  the  needle-track,  is 
moderately  dense,  is  accompanied  by  the  formation  of 
gas-bubbles,  and  the  surrounding  gelatin  is  more  or 
less  clear. 

These  distinctions  were  found  by  Hiss  to  be  much 
more  marked  with  the  slowly  or  feebly  motile  speci- 
mens of  the  colon  bacillus  than  when  the  actively  motile 
varieties  were  used.  Many  of  these  latter,  except  for 
their  power  to  ferment  glucose  with  liberation  of  gas, 
were  almost  indistinguishable  from  the  typhoid  bacillus 
in  so  far  as  their  power  to  wander  through  and  cloud 
the  medium  was  concerned.  For  the  isolation  and  dif- 
ferentiation of  colonies  of  the  two  organisms  by  the 
plate  method  the  following  medium  was  employed : 

Agar-agar 10  grammes. 

Gelatin .   ........      25        " 

Liebig's  beef-extract .'....        5        " 

Sodium  chloride 5        " 

Glucose     .....   .t. 10        " 

Water 1000  c.c. 

The  reaction  of  this  medium  is  equivalent  to  2  per  cent, 
of  normal  acid  to  the  litre — i.  e.,  an  acidity  that  would 
require  20  c.c.  of  normal  sodium  hydroxide  solution  to 
the  litre  to  bring  it  to  the  phenolphtalein  neutral  point. 
In  plates  made  from  this  medium  the  deep  colonies  of 
bacillm  typhosus  are  small,  more  or  less  spherical,  and 


390  BACTERIOLOGY. 

have  a  rough,  irregular  outline.  Their  most  character- 
istic feature  "  consists  of  well-defined,  filamentous  out- 
growths, ranging  from  a  single  thread  to  a  complete 
fringe  around  the  colony.  The  young  colonies  are  at 
times  composed  solely  of  threads."  The  fringing 
threads  grow  almost  straight  out  from  the  colonies. 
The  surface  colonies  are  small  and  have  usually  a 
dense  centre  that  is  surrounded  by  an  almost  trans- 
parent zone  or  by  a  fringe  of  threads  somewhat  similar 
to  those  seen  about  the  deeper  colonies. 

The  deep  colonies  of  the  colon  bacillus  are,  as  a  rule, 
larger,  denser,  of  an  oval  or  lens-shape,  and  are  more 
sharply  circumscribed  than  those  of  bacillus  typhosus. 
On  the  surface  they  are  also  larger,  and,  as  a  rule, 
spread  out  as  a  moderately  thick  layer  from  a  denser 
centre.  The  younger  the  colonies  of  the  typhoid  bacil- 
lus the  more  characteristic  their  appearance.  They  are 
seen  at  their  best  after  from  16  to  18  hours7  growth  at 
37.5°  C.1 

Method  of  Capaldi  and,  Proskauer.2  As  a  result  of 
an  elaborate  series  of  experiments,  these  authors  recom- 
mend the  use  of  two  special  culture-media  for  the  dif- 
ferentiation of  the  typhoid  and  colon  bacilli. 

Medium  No.  1  consists  of: 

Asparagine 0.2    per  cent,  in  distilled  water. 

Mannite 0.2  '  " 

Sodium  chloride 0.02  " 

Magnesium  sulphate    ....  0.01  " 

Calcium  chloride 0.02  '  " 

Mono-potassium  phosphate    .  0.2 

1  The  reader  is  referred  to  the  original  article  for  many  important 
details  that  are  not  included  here. 

2  Capaldi  and  Proskauer :  Zeitschrift  fur  Hygiene  und  Infektions- 
krankheiten,  1896,  Band  xxiii.  S.  452. 


ISOLATING  THE  TYPHOID  BACILLUS.         391 
Medium  No.  2  consists  of: 

Witte's  peptone 2.0  per  cent,  in  distilled  water. 

Mannite 0.1 

Both  media  are  to  be  sterilized,  the  reaction  brought 
to  the  litmus  neutral  point  with  caustic  potash  solu- 
tion, and  enough  litmus  tincture  then  added  to  them  to 
cause  a  distinct,  though  not  too  intense,  purple  color. 
After  this  they  are  to  be  again  sterilized,  when  they 
are  ready  for  use. 

After  20  hours  at  37°-38°C.  typical  colon  bacilli 
and  the  varieties  of  this  organism  develop  in  both  solu- 
tions, but  produce  add  only  in  medium  No.  1. 

The  typhoid  bacillus  grows  only  in  medium  No.  2, 
and  its  growth  is  accompanied  by  the  production  of 
an  acid  reaction. 

The  growth  of  bacillus  coli  in  medium  No.  2  is  ac- 
companied by  a  slight  alkaline  reaction. 

The  alterations  in  reaction  are  indicated  by  the  cor- 
responding changes  in  the  color  of  the  litmus  tincture 
in  the  media. 

It  is  interesting  to  note  that  in  this  test  the  usual 
reactions  produced  by  these  organisms  in  peptone  media 
containing  the  ordinary  fermentable  carbohydrates,  such 
as  glucose  and  lactose,  are  reversed. 

The  authors  state  that  this  method  has  thus  far  shown 
itself  to  be  infallible  for  the  differentiation  of  cultures 
of  typhoid  and  colon  bacilli  obtained  by  them  from 
ever)1  available  source. 

The  spleen  of  a  patient  dead  of  typhoid  fever  is  the 
most  reliable  source  from  which  to  obtain  cultures  of  the 
typhoid  bacillus  for  study.  But  it  must  always  be  re- 
membered that  the  same  channels  through  which  the 


392  BACTERIOLOGY. 

typhoid  bacillus  gains  access  to  this  viscus  are  like- 
wise open  to  other  organisms  present  in  the  intestines, 
and  for  this  reason  bacterium  coli  commune,  a  normal 
inhabitant  of  the  colon,  may  also  be  found  in  this 
locality. 

NOTE. — Obtain  a  pure  culture  of  typhoid  bacilli,  and 
from  this  make  inoculations  upon  a  series  of  potatoes 
of  different  ages  and  from  different  sources.  Do  they 
all  grow  alike  ? 

Before  sterilizing  render  another  lot  of  potatoes  slightly 
acid  with  a  few  drops  of  very  dilute  acetic  acid ;  render 
others  very  slightly  alkaline  with  dilute  caustic  soda. 
Are  any  differences  in  the  growths  noticeable  ? 

Make  a  series  of  twelve  tubes  of  peptone  solution  to 
which  rosolic  acid  has  been  added.  Inoculate  them  all 
with  as  nearly  the  same  amount  of  material  as  possible 
(one  loopful  from  a  bouillon  culture  into  each  tube) ; 
place  them  all  in  the  incubator.  Is  the  color-change, 
as  compared  with  that  of  the  control-tube,  the  same  in 
all  cases. 

Compare  the  morphology  of  cultures  of  the  same  age 
on  gelatin,  agar-agar,  and  potato. 

Select  a  culture  in  which  the  vacuolations  are  quite 
marked.  Examine  this  culture  unstained.  Do  the 
organisms  look  as  if  they  contained  spores?  How 
would  you  demonstrate  that  the  vacuolations  are  not 
spores  ?  What  is  the  crucial  test  for  spores  ? 

Obtain  from  normal  faeces  a  pure  culture  of  the 
commonest  organism  present.  Write  a  full  description 
of  it.  Now  make  parallel  cultures  of  this  organism  and 
of  the  typhoid  bacillus  on  all  the  different  media.  How 
do  they  differ  ?  In  what  respects  are  they  similar  ? 


BACTERIUM  COLI  COMMUNE.  393 

BACTERIUM  COLI  COMMUNE  ;  BACILLUS  COLI  COMMUNIS 
COLON  BACILLUS;  BACILLUS  NEAPOLITANUS  OF 
EMMERICH. 

This  organism  was  discovered  by  Escherich,  in  1885, 
in  the  intestinal  discharges  of  milk-fed  infants.  It  has 
since  been  demonstrated  to  be  a  normal  inhabitant  of  the 
intestines  of  man  and  of  certain  domestic  animals 
(bo vines,  hogs,  dogs). 

For  a  time  after  its  discovery  it  was  considered  of 
but  little  importance  and  attracted  attention  only  be- 
cause of  its  resemblance,  in  certain  respects,  to  the  bacil- 
lus of  typhoid  fever,  with  which  it  was  occasionally 
confounded.  In  this  particular  it  still  serves  as  a 
subject  for  study.  Some  have  even  gone  so  far  as  to 
regard  them  as  but  varieties  of  one  and  the  same 
species,  though  in  the  present  state  of  our  knowledge 
this  is  an  assumption  for  which  as  yet  there  are 
not  sufficient  grounds.  That  they  possess  in  common 
certain  general  points  of  resemblance  and  often  ap- 
proach one  another  in  some  of  their  biological  peculiar- 
ities is  true ;  but,  as  we  shall  learn,  they  each  possess 
peculiarities  which,  when  considered  together,  render 
their  differentiation  from  one  another  a  matter  of  but 
little  difficulty. 

With  theAvider  application  of  bacteriological  methods 
to  the  study  of  pathological  processes  it  was  occasionally 
observed  that,  under  favorable  circumstances,  bacilla* 
coll  communis  disseminated  from  its  normal  habitat 
and  appeared  in  remote  organs,  often  associated  with 
diseased  conditions.  This  was  at  first  considered 
of  but  little  importance,  and  its  presence  in  these 
localities  was  usually  regarded  as  accidental.  Its 


394  BACTERIOLOGY. 

repeated  appearance,  however,  in  different  parts  of 
the  body  outside  of  the  intestines,  and  the  frequency 
of  its  association  with  pathological  conditions,  ultimately 
attracted  attention  to  it,  and  in  consequence  during  the 
past  few  years  a  great  deal  has  been  written  concerning 
the  possible  pathogenic  nature  of  this  organism. 

The  fact  that  it  is  a  commensal  species,  always  inti- 
mately associated  with  certain  of  our  life-processes, 
together  with  the  fact  that  it  is  known  to  appear  in 
organs  other  than  that  in  which  it  is  normally  located, 
and  that  its  occurrence  in  diseased  conditions  is  not 
rare,  justifies  the  opinion  that  it  is  one  of  the  most 
important  of  the  micro-organisms  with  which  we  have 
to  deal. 

While  not  generally  considered  a  pathogenic  organ- 
ism, there  is,  nevertheless,  sufficient  evidence  to  war- 
rant the  statement  that,  under  favorable  conditions, 
with  which  we  are  not  entirely  familiar,  this  organism 
may  assume  pathogenic  properties,  so  that  its  presence 
in  diseased  conditions  is  not  always  to  be  considered  as 
accidental,  though  this  is  frequently  the  case. 

The  morphological  and  cultural  peculiarities  of  bac- 
terium coli  commune  are  as  follows  : 

MORPHOLOGY. — In  shape  it  is  a  rod  with  rounded 
ends,  sometimes  so  short  as  to  appear  almost  spherical, 
while  again  it  is  seen  as  very  much  longer  threads. 
Often  both  forms  are  associated  in  the  same  culture. 
It  may  occur  as  single  cells,  or  as  pairs  joined  end  to 
end. 

It  has  no  peculiar  morphological  features  that  can 
aid  in  its  identification,  for  in  this  respect  it  simu- 
lates many  other  organisms.  It  is  usually  said  to  be 
motile,  and  undoubtedly  is  motile  in  the  majority  of 


BACTERIUM  COLT  COMMUNE.  395 

cases ;  but  its  movements  are  at  time  so  sluggish  that  a 
positive  opinion  is  often  difficult. 

By  Loffler's  method  of  staining,  flagella  can  be  de- 
monstrated, though  usually  not  in  such  numbers  as  are 
seen  to  occur  on  the  typhoid  fever  bacillus. 

It  does  not  form  spores. 

It  grows  both  with  and  without  free  oxygen. 

Ox  GELATIN. — On  the  surface  its  colonies  appear  as 
small,  dry,  irregular,  flat,  blue-white  points  that  are 
commonly  somewhat  dentated  or  notched  at  the  margin. 
They  are  a  trifle  denser  at  the  centre  than  at  the 
periphery,  and  are  often  marked  at  or  near  the  middle 
by  an  oval  or  round  nucleus-like  mass — the  original 
colony  from  which  the  layer  on  the  surface  developed. 
When  located  in  the  depths  of  the  gelatin,  and  ex- 
amined with  a  low-power  lens,  they  are  at  first  seen  to 
be  finely  granular  and  of  a  very  pale  greenish-yellow 
color;  later  they  become  denser,  darker,  and  much 
more  markedly  granular;  in  shape  they  are  round, 
oval,  and  lozenge-like.  When  the  surface  colonies  are 
viewed  under  a  low  power  of  the  microscope  they  pre- 
sent essentially  the  same  appearance  as  that  given  for 
the  colonies  of  the  bacillus  of  typhoid  fever,  viz.,  they 
resemble  flattened  pellicles  of  glass-wool,  or  patches  of 
finely  ground  colorless  glass.  Colonies  of  this  organ- 
ism on  gelatin  are  frequently  encountered  that  cannot 
be  distinguished  from  those  resulting  from  the  growth 
of  bacillus  typhosm  ;  though,  as  a  rule,  their  growth  is  a 
little  more  luxuriant. 

In  stab-  and  smear-cultures  on  gelatin  the  surface- 
growth  is  flat,  dry,  and  blue-white  or  pearl  color. 
Limited  growth  occurs  along  the  track  of  the  needle  in 
the  depths  of  the  gelatin.  As  the  culture  becomes 


396  BACTERIOLOGY. 

older  the  gelatin  round  about  the  surface-growth  may 
gradually  lose  its  transparency  and  become  cloudy, 
often  quite  opaque.  In  still  older  cultures  small  root- 
or  branch-like  projections  from  the  surface-growth  into 
the  gelatin  are  sometimes  seen.  At  times  these  may  be 
of  a  distinctly  crystalline  appearance. 

It  does  not  cause  liquefaction  of  gelatin. 

Its  growth  on  nutrient  agar-agar  and  on  blood- serum 
is  luxuriant,  but  not  characteristic. 

In  bouillon  it  causes  diffuse  clouding  with  sedimen- 
tation. In  some  bouillon  cultures  an  attempt  at  pel- 
licle-formation on  the  surface  may  be  seen,  but  this 
is  exceptional.  In  old  bouillon  cultures  the  reaction 
becomes  alkaline  and  a  decided  faecal  odor  may  be 
detected. 

Its  growth  on  potato  is  rapid  and  voluminous,  ap- 
pearing after  twenty-four  to  thirty-six  hours  in  the 
incubator  as  a  more  or  less  lobulated  layer  of  a  drab, 
dark-cream,  or  brownish-yellow  color. 

In  neutral  milk  containing  a  little  litmus  tincture 
the  blue  color  is  changed  to  red  after  from  eighteen  to 
twenty-four  hours  in  the  incubator,  and,  in  addition? 
the  majority  of  cultures  cause  firm  coagulation  of  the 
casein  in  about  thirty-six  hours,  though  frequently  this 
takes  longer.  Very  rarely  the  litmus  may  indicate  the 
production  of  acid  and  no  coagulation  occur. 

In  media  containing  glucose  it  grows  rapidly  and 
causes  active  fermentation,  with  liberation  of  carbonic 
acid  and  hydrogen.  If  cultivated  in  solid  media  to 
which  glucose  (2  per  cent.)  has  been  added,  the  gas- 
formation  is  recognized  by  the  appearance  of  numerous 
bubbles  along  and  about  the  points  of  growth.  If  cul- 
tivated in  fluid  media,  also  containing  glucose,  in  the 


BACTERIUM  COLI  COMMUNE.  397 

fermentation-tube,  evidence  of  fermentation  is  given  by 
the  collection  of  gas  in  the  closed  arm  of  the  tube. 

On  lactose-litmus-agar-agar  its  colonies  are  pink  and 
the  color  of  the  surrounding  medium  is  changed  from 
1)1  ue  to  red. 

It  produces  indol  in  both  bouillon  and  peptone  solu- 
tion. 

In  Dunham's  peptone  solution  it  produces  indol  in 
from  forty-eight  to  seventy-two  hours. 

It  stains  with  the  ordinary  aniline  dyes.  It  is  decol- 
orized when  treated  by  the  method  of  Gram. 

By  comparing  what  has  been  said  of  bacillus  typho- 
sus  and  of  bacterium  coli  commune  it  will  be  seen  that 
while  they  simulate  each  other  in  certain  respects,  they 
nevertheless  possess  individual  characteristics  by  which 
they  may  readily  be  differentiated.  The  least  variable 
of  the  differential  points  are  : 

1.  Motility  of  bacillus  typhosus  is  much  more  con- 
spicuous, as  a  rule,  than  is  that  of  bacterium  coli  com- 
mune. 

2.  On  gelatin  colonies  of  the  typhoid  bacillus  de- 
velop more  slowly  than  do  those  of  the  colon  bacillus. 

3.  On  potato  the  growth  of  the.  typhoid  bacillus  is 
usually  invisible  (though  not  always) ;  while  that  of  the 
colon  bacillus  is  rapid,  luxuriant,  and  always  visible. 

4.  The  typhoid  bacillus  does  not  cause  coagulation  of 
milk  with  acid  reaction.     The  colon  bacillus  does  this 
in  from  thirty-six  to  forty-eight  hours  in  the  incubator. 

5.  The  typhoid  bacillus  never  causes  fermentation, 
with  liberation  of  gas,  in  media  containing  glucose,  lac- 
tose, or  saccharose.     The  colon  bacillus  is  conspicuous 
for  its  power  of  causing  gaseous  fermentation  in  such 
solutions. 


398  BACTERIOLOGY. 

6.  In  nutrient  agar-agar  or  gelatin  containing  lactose 
and  litmus  tincture,  and  of  a  slightly  alkaline  reaction, 
the  color  of  the  colonies  of  typhoid  bacillus  is  pale  blue, 
and  there  is  no  reddening  of  the  surrounding  medium ; 
while  colonies  of  the  colon  bacillus  are  pink  and  the 
medium  round  about  them  becomes  red. 

7.  The  typhoid  bacillus  does  not,  as  a  rule,  possess  the 
property  of  producing  indol  in  solutions  of  peptone ;  the 
growth  of  the  colon  bacillus  in  these  solutions  is  accom- 
panied by  the  production  of  indol  in  from  forty-eight 
to  seventy-two  hours  at  37°  to  38°  C. 

ANIMAL  INOCULATIONS. — As  with  the  bacillus  of 
typhoid  fever,  the  results  of  inoculation  of  animals  with 
cultures  of  this  organism  cannot  be  safely  predicted. 
According  to  the  observations  of  Escherich,  Emmerich, 
Weisser,  and  others,  the  effects  that  do  appear  are  in 
most  instances  to  be  attributed  to  the  toxic  rather  than 
to  the  infective  properties  of  the  culture  used. 

When  introduced  into  the  subcutaneous  tissues  of 
mice  it  has  no  effect,  while  similar  inoculations  of  guinea- 
pigs  are  sometimes  (not  always)  followed  by  abscess- 
formation  at  the  point  of  operation,  or  by  alterations  very 
similar  to  those  produced  by  intravascular  inoculation, 
viz.,  death  in  less  than  twenty-four  hours,  accompanied 
by  redness  of  the  peritoneum  and  marked  hypersemia 
and  ecchymoses  of  the  small  intestine,  together  with 
swelling  of  Peyer's  patches.  The  caecum  and  colon 
may  remain  unchanged  or  present  enlarged  follicles. 
There  may  or  may  not  be  an  accumulation  of  fluid  in 
the  abdominal  cavity ;  but  peritonitis  is  rarely  present. 
The  small  intestine  may  contain  bloody  mucus. 

Intravenous  inoculation  of  rabbits  may  be  followed 
by  similar  changes,  with  often  the  occurrence  of  diar- 


BACTERIUM  COLI  COMMUNE.  399 

rhoea  before  death,  which  may,  in  the  acute  cases,  result 
in  from  three  to  forty  hours.  In  another  group  of 
cases  acute  fatal  intoxication  does  not  result,  and  the 
animal  lives  for  weeks  or  months,  dying  ultimately  of 
what  appears  to  be  the  effects  of  a  slow  or  chronic  form 
of  infection.  For  a  few  hours  after  inoculation  these 
animals  present  no  marked  symptoms;  exceptionally, 
somnolence  and  diarrhoea  have  been  observed  at  this 
period,  indicating  acute  intoxication  from  which  the 
animal  lias  recovered.  The  affection  is  unattended  by 
fever.  The  most  marked  symptom  is  loss  of  weight. 
This  is  usually  progressive  from  the  first  or  second  day 
after  inoculation,  with  slight  fluctuations  until  death. 

At  autopsy  the  animal  is  found  to  be  emaciated. 
The  subcutaneous  tissues  and  the  muscles  appear  pale 
and  dry.  The  serous  cavities,  particularly  the  pericar- 
dial,  may  contain  an  excess  of  serum.  The  viscera  are 
anaemic.  The  spleen  is  small,  thin,  and  pale.  Ex- 
ceptionally ulcers  and  ecchymoses  are  observed  in  the 
caecum,  but  generally  there  are  no  lesions  of  the  intes- 
tinal tract. 

The  most  striking  and  constant  lesions,  those  most 
characteristic  of  the  affection,  are  in  the  bile  and  in  the 
liver ;  in  some  cases  the  quantity  of  bile  may  not  exceed 
the  normal,  but  in  others  the  gall-bladder  maybe  ab- 
normally distended  with  bile.  The  bile  is  nearly  color- 
less or  has  a  pale  yellowish  or  brownish  tint,  with  little 
or  no  greenish  color.  Its  consistence  is  much  less 
viscid  than  normal,  being  often  thin  and  watery.  It 
usually  contains  small,  opaque,  yellowish  particles  or 
clumps  which  can  be  seen  floating  in  it,  even  through  the 
walls  of  the  gall-bladder.  These  clumps  consist  micro- 
scopically of  bile-stained,  apparently  necrotic,  epithelial 


400  BACTERIOLOGY. 

cells ;  leucocytes  in  small  numbers ;  amorphous  masses 
of  bile-pigment,  and  bacteria  often  in  zooglrea-like 
clumps.  Similar  material  is  found  in  the  larger  bile- 
ducts. 

The  liver  frequently  contains  opaque,  whitish  or  yel- 
lowish-white spots  and  streaks  of  irregular  size  and 
shape,  which  give  a  peculiar  mottling  to  the  organ  when 
present  in  large  number.  These  areas  may  be  numer- 
ous, or  only  one  or  two  may  be  found.  In  size  they 
range  from  minute  points  to  areas  of  from  2  to  3  cm.  in 
extent.  By  microscopic  examination  they  are  found  to 
represent  localities  where  the  liver-cells  have  undergone 
necrosis  accompanied  by  emigration  of  leucocytes,  and 
the  cells  about  them  are  in  a  condition  of  fatty  degenera- 
tion. In  sections  of  the  liver  masses  of  the  bacilli  may 
be  discovered  in  and  about  the  necrotic  foci  just  de- 
scribed. 

At  these  autopsies  the  colon  bacillus  is  not  found 
generally  distributed  through  the  body,  but  is  only  to 
be  detected  in  the  bile,  liver,  and  occasionally  in  the 
spleen.1 

1  Consult  paper  by  Blachstein  on  this  subject,  Johns  Hopkins  Hos- 
pital Bulletin,  1891,  vol.  ii.,  p.  96. 


CHAPTER    XXII. 

The  spirillum  (comma  bacillus)  of  Asiatic  cholera — Its  morphological 
and  cultural  peculiarities — Pathogenic  properties — The  bacterio- 
logical diagnosis  of  Asiatic  cholera. 

At  the  conference  held  in  Berlin  in  1884  for  the  pur- 
pose of  discussing  Asiatic  cholera  from  the  sanitary 
aspect,  it  was  announced  by  Koch1  that  he  had  dis- 
covered in  the  intestinal  evacuations  of  individuals  suf- 
fering from  Asiatic  cholera  a  micro-organism  that  he 
believed  to  be  the  cause  of  the  malady.  The  importance 
of  this  statement  necessarily  attracted  widespread  atten- 
tion to  the  subject,  and  as  one  of  the  consequences  there 
existed,  for  a  short  time  following,  some  skepticism  as 
to  the  accuracy  of  Koch's  claim.  These  doubts  arose  as 
a  result  of  a  series  of  contributions  from  other  observers, 
who  endeavored  to  prove  that  the  organism  found  by 
Koch  in  cholera  evacuations  was  common  to  other  local- 
ities, and  was  not  a  specific  accompaniment  of  this  dis- 
ease. It  was  not  very  long,  however,  before  it  was 
evident  that  these  objections  were  based  upon  untrust- 
worthy observations,  and  that  by  reliable  methods  of 
investigation  the  organism  to  which  he  had  called  atten- 
tion could  be  easily  differentiated  from  each  of  those 
with  which  it  was  claimed  to  be  identical. 

This  organism,  known  both  as  the  spirillum  of  Asiatic 
cholera,  and,  because  of  its  morphology,  as  Koch's 

1  Verhandlungen  der  Conferenz  zur  Erorterung  der  Cholerafrage. 
1884,  Berlin. 

26  401 


402  BACTERIOLOGY. 

"  comma  bacillus/7  is  identified  by  the  following  peculi- 
arities : 

SPIRILLUM    CHOLERA   ASIATICS. 

MORPHOLOGY. — It  is  a  slightly  curved  rod,  ranging 
from  about  0.8  to  2  //in  length  and  from  0.3  to  0.4  p. 
in  thickness — that  is  to  say,  it  is  usually  from  about  one- 
half  to  two-thirds  the  length  of  the  tubercle  bacillus, 
but  is  thicker  and  plumper.  Its  curve  is  frequently 
not  more  marked  than  that  of  a  comma,  and,  indeed,  it 
is  often  almost  straight ;  at  times,  though,  the  curve  is 
much  more  pronounced,  and  may  even  describe  a  semi- 
circle. Occasionally  the  curve  may  be  double,  one 
comma  joining  another,  with  their  convexities  pointing 
in  opposite  directions,  so  that  a  figure  similar  to  the 
letter  S  is  produced.  In  cultures  long  spiral  or  undu- 
lating threads  may  often  be  seen.  From  these  appear- 
ances this  organism  cannot  be  considered  as  a  bacillus, 
but  rather  as  an  intermediate  type  between  the  bacilli 
and  the  spirilla.  Koch  thinks  it  not  improbable  that 
the  short  comma  forms  represent  segments  of  a  true 
spirillum,  the  normal  form  of  the  organism.  (Fig.  73.) 

It  does  not  form  spores,  and  we  have  no  reliable  evi- 
dence that  it  possesses  the  property  of  entering,  at  any 
time,  a  stage  in  which  its  powers  of  resistance  to  detri- 
mental agencies  are  increased. 

It  is  a  flagellated  organism,  but  has  only  a  single 
flagellum  attached  to  one  of  its  ends. 

It  is  actively  motile,  especially  in  the  comma  stage ; 
though  the  long  spiral  forms  also  possess  this  property. 

GROUPING. — As  found  in  the  slimy  flakes  in  the  intes- 
tinal discharges  from  cholera  patients,  Koch  likens  its 
mode  of  grouping  to  that  seen  in  a  school  of  small  fish 


SPIRILLUM  CHOLERA  ASIATICS.  403 

when  swimming  up  stream— i.  e.,  they  all  point  in  nearly 
the  same  direction,  and  lie  in  irregularly  parallel,  linear 


FIG.  73. 


Spirillum  of  Asiatic  cholera.    Impression  cover-slip  from  a  colony  thirty- 
four  hours  old. 

groups  that   are  formed  by  one  comma  being  behind 
the  other  without  being  attached  to  it. 


FIG.  74. 


t 


\     f 

f 


Involution-forms  of  the  spirillum  of  Asiatic  cholera,  as  seen  in  old  cultures. 

On  cover-slip  preparations  made  from  cultures  in  the 
ordinary  way  there  is  nothing  characteristic  about  the 
grouping  ;  but  in  impression  cover-slips  made  from 
young  cultures  the  short  commas  will  nearly  always  be 
seen  in  small  groups  of  three  or  four,  lying  together  in 
such  a  way  as  to  have  their  long  axes  nearly  parallel  to 
one  another.  (See  Fig.  73.) 

In  old  cultures  in  which  development  has  ceased  it 
undergoes  degenerative  changes,  and  the  characteristic 


404  BACTERIOLOGY. 

comma  and  spiral  shapes  may  entirely  disappear,  their 
place  being  taken  by  irregular  involution-forms  that 
present  every  variety  of  outline.  (See  Fig.  74.)  In 
this  stage  they  take  on  the  stain  very  feebly,  and  often 
not  at  all. 

CULTURAL  PECULIARITIES. — On  plates  of  nutrient 
gelatin  that  have  been  prepared  from  a  pure  culture  of 
this  organism  and  kept  at  a  temperature  of  from  20° 
to  22°  C.,  development  can  often  be  observed  after  as 
short  a  period  as  twelve  hours,  but  frequently  not  be- 
fore sixteen  to  eighteen  hours.  This  is  especially  true 
of  the  first  or  "  original "  plate,  containing  the  largest 
number  of  colonies.  At  this  time  the  plate  will  pre- 
sent to  the  naked  eye  an  appearance  that  has  been 
likened  to  a  ground-glass  surface,  or  to  a  surface  that 
has  been  stippled  with  a  finely  pointed  needle,  or  one 
upon  which  very  fine  dust  has  been  sprinkled.  This 
appearance  is  due  to  the  presence  of  minute  colonies 
closely  packed  together  upon  the  surface  of  the  gelatin. 
In  the  depth  of  the  gelatin  can  also  be  seen  closely 
packed,  small  points,  likewise  representing  growing 
colonies.  As  growth  progresses  liquefaction  occurs 
around  the  superficial  colonies,  and  in  consequence  this 
plate  is  usually  entirely  liquid  after  from  twenty-four 
to  thirty  hours ;  the  developmental  phases  through  which 
the  colonies  pass  cannot,  therefore,  be  studied  upon  it. 

On  plates  2  and  3,  where  the  colonies  are  more  widely 
separated,  they  can  be  seen  after  twenty-four  to  thirty 
hours  as  small,  round  or  oval,  white  or  cream-white 
points,  and  when  located  superficially  a  narrow  trans- 
parent zone  of  liquefaction  can  be  detected  around 
them.  As  growth  continues  this  liquefaction  extends 
downward  rather  than  laterally,  and  the  colony  ulti- 


SPIRILLUM  CHOLERA  ASIATICS. 


405 


mately  assumes  the  appearance  of  a  dense,  white  mass 
lying  at  the  bottom  of  a  sharply-cut  pit  or  funnel  con- 
taining transparent  fluid.  This  liquefaction  is  never 
very  widespread  nor  rapid,  and  rarely  extends  more 
than  one  millimetre  beyond  the  colony  proper.  On 
plates  containing  few  colonies  there  is  little  or  no 
tendency  for  them  to  become  confluent,  and  they  rarely 
exceed  2  to  3  mm.  in  diameter. 


FIG.  75. 


- 

a 


c  d 

Developmental  phases  of  colonies  of  the  spirillum  of  Asiatic  cholera  at 

20°  to  22°  C.  on  gelatin.    X  about  75  diameters. 

a.  After  sixteen  to  eighteen  hours.  6.  After  twenty-four  to  twenty-six 
hours,  'c.  After  thirty-eight  to  forty  hours,  d.  After  forty-eight  to  fifty 
hours,  e.  After  sixty-four  to  seventy  hours. 

When  examined  tinder  a  low  magnifying  lens  the  very 
young  colonies  (sixteen  to  eighteen  hours  old)  appear 
as  pale,  translucent,  granular  globules  of  a  very  delicate 
greenish  or  yellowish-green  color,  sharply  outlined,  and 
not  perfectly  round.  (See  a,  Fig.  75.)  As  growth  p^o- 
gresses  this  homogeneous  granular  appearance  is  re- 
placed by  an  irregular  lobulation,  and  ultimately  the 
sharply-cut  margin  of  the. colony  becomes  dentated  or 
scalloped.  (See  b  and  c,  Fig.  75.)  After  forty-eight 
hours  (and  frequently  sooner)  liquefaction  of  the  gelatin 


406  BACTERIOLOGY. 

has  taken  place  to  such  an  extent  that  the  appearance 
of  the  colony  is  entirely  altered.  Under  a  magnify- 
ing glass  the  colony  proper  is  now  seen  to  be  ragged 
about  its  edges,  while  here  and  there  shreds  of  the 
colony  can  be  detected  scattered  through  the  liquid 
into  which  it  is  sinking.  These  shreds  evidently  repre- 
sent portions  of  the  colony  that  became  detached  from 
its  margin  as  it  gradually  sank  into  the  liquefied  area. 

At  d,  in  Fig.  75,  is  seen  a  representation  of  the 
several  appearances  afforded  by  the  colonies  at  this 
stage.  At  the  end  of  the  second,  or  during  the  early 
part  of  the  third  day,  the  sinking  of  the  colonies  into 
the  liquefied  pits  resulting  from  their  growth  is  about 
complete,  and  under  a  low-power  lens  they  now  appear  as 
dense,  granular  masses,  surrounded  by  an  area  of  lique- 
faction through  which  can  be  seen  granular  prolonga- 
tions of  the  colony,  usually  extending  irregularly  be- 
tween the  periphery  and  the  central  mass.  (See  e,  Fig. 
75.)  If  the  periphery  be  examined,  it  will  be  seen  to 
be  fringed  with  delicate,  cilia-like  lines  that  radiate 
from  it  in  much  the  same  way  that  cilia  radiate  from 
the  ends  of  the  columnar  epithelial  cells  lining  the  air- 
passages. 

These  are  the  more  marked  phases  through  which  the 
colonies  of  this  organism  pass  in  their  development  on 
gelatin  plates.  In  some  cultures  the  various  phases 
here  given  pass  in  succession  more  quickly,  while  in 
cultures  from  other  sources  they  may  be  somewhat  re- 
tarded. 

On  plates  of  nutrient  agar-agar  the  appearance  of  the 
colonies  is  not  characteristic.  They  appear  as  round  or 
oval  patches  of  growth  that  are  moist  and  moderately 
transparent.  The  colonies  on  this  medium  at  37°  C. 


SPIRILLUM  CHOLERJE  ASIATICS. 


407 


naturally  grow  to  a  larger   size  than  do  those   upon 
gelatin  at  22°  C. 

In  stab-cultures  in  gelatin  there  appears  at  the  top 
of  the  needle-track  after  thirty-six  to  forty-eight  hours 
at  22°  C.  a  small,  funnel-shaped  depression.  As  the 
growth  progresses  liquefaction  occurs  about  this  point. 
In  the  centre  of  the  depression  can  be  distinguished 


FIG.  76. 


I 


abed 
Stab-culture  of  the  spirillum  of  Asiatic  cholera  in  gelatin, 

at  18°  to  20°  C. 

a.  After  twenty-four  hours,    b.  After  forty-eight  hours,    c.  After  seventy- 
two  hours,    d.  After  ninety-six  hours. 

a  small,  dense,  whitish  clump,  the  colony  itself.  As 
growth  continues  the  depression  increases  in  extent 
and  ultimately  assumes  an  appearance  that  consists  in 


408  BACTERIOLOGY. 

the  apparent  sinking  of  the  liquefied  portion  in  such 
a  way  as  to  leave  a  perceptible  air-space  between  the 
top  of  the  liquid  and  the  surface  of  the  solid  gelatin. 
The  growth  now  appears  to  be  capped  by  a  small  air- 
bubble.  The  impression  given  by  it  at  this  stage  is  not 
only  that  there  has  been  a  liquefaction,  but  also  a  coin- 
cident evaporation  of  the  fluid  from  the  liquefied  area 
and  a  constriction  of  the  superficial  opening  of  the  fun- 
nel. (See  a,  b,  c,  and  d,  Fig.  76.)  Liquefaction  is  not 
especially  active  along  the  deeper  portions  of  the  track 
made  by  the  needle,  though  in  stab-cultures  in  gelatin  the 
liquefaction  is  much  more  extensive  than  that  usually 
seen  around  colonies  on  plates.  It  spreads  laterally  at 
the  upper  portion,  and  after  about  a  week  a  large  part 
of  the  gelatin  in  the  tube  may  have  become  fluid,  and 
the  growth  will  have  lost  its  characteristic  appearance. 

Stab-  and  smear-cultures  on  agar-agar  present  noth- 
ing characteristic. 

Its  growth  in  bouillon  is  luxuriant,  causing  a  diffuse 
clouding  and  the  ultimate  production  of  a  delicate  film 
upon  the  surface. 

In  sterilized  milk  of  a  neutral  or  amphoteric  reaction 
at  a  temperature  of  36°  to  38°  C.  it  develops  actively, 
and  gradually  produces  an  acid  reaction,  with  coagula- 
tion of  the  casein.  It  retains  its  vitality  under  these 
conditions  for  about  three  weeks  or  more.  The  blue 
color  of  milk  to  which  neutral  litmus  tincture  has  been 
added  is  changed  to  pink  after  thirty-six  or  forty-eight 
hours  at  body-temperature. 

Its  growth  in  peptone  solution,  either  that  of  Dun- 
ham (see  Special  Media)  or  the  one  preferred  by  Koch, 
viz.,  2  parts  of  Witte's  peptone,  1  part  of  sodium  chlo- 
ride, and  100  parts  of  distilled  water,  is  accompanied  by 


SPIRILLUM  CHOLERA  ASIATICS  409 

the  production  of  both  indol  and  nitrites,  so  that  after 
eight  to  twelve  hours  in  the  incubator  at  37°  C.  the  rose 
color  characteristic  of  indol  appears  upon  the  addition 
of  sulphuric  acid  alone.  (See  Indol  Reaction.) 

(What  does  the  presence  of  nitrites  in  these  cultures 
signify?) 

In  peptone  solution  to  which  rosolic  acid  has  been 
added  the  red  color  is  very  much  intensified  after  four 
or  five  days  at  37°  C. 

Its  growth  on  potato  of  a  slightly  acid  reaction  is  seen 
after  three  or  four  days  at  37°  C.  as  a  dull,  whitish, 
non-glistening  patch  at  and  about  the  site  of  inocula- 
tion. It  is  not  elevated  above  the  surface  of  the  potato, 
and  can  only  be  distinctly  seen  when  held  to  the  light 
in  a  particular  position.  Growth  on  acid  potato  occurs, 
however,  only  at  or  near  the  body-temperature,  owing 
probably  to  the  acid  reaction,  which  is  sufficient  to  pre- 
vent development  at  a  lower  temperature,  but  does  not 
have  this  effect  when  the  temperature  is  more  favorable. 
On  solidified  blood-serum  growth  is  usually  said  to 
be  accompanied  by  slow  liquefaction.  I  have  not  suc- 
ceeded in  obtaining  this  result  on  Loftier' s  serum,  nor 
have  I  detected  anything  characteristic  about  its  growth 
on  this  medium. 

The  temperature  most  favorable  for  its  growth  is 
between  35°  and  38°  C.  It  grows,  but  more  slowly, 
at  17°  C.  Below  16°  C.  no  growth  is  visible. 

It  is  not  destroyed  by  freezing.  When  exposed  to 
65°  C.  its  vitality  is  destroyed  in  five  minutes. 

It  is  strictly  aerobic,  its  development  ceasing  if  the 
supply  of  oxygen  be  cut  off. 

It  does  not  grow  in  an  atmosphere  of  carbonic  acid, 
but  is  not  killed  by  a  temporary  exposure  to  this  gas. 


410  BACTERIOLOGY. 

It  does  not  grow  in  acid  media,  but  flourishes  best  in 
media  of  neutral  or  slightly  alkaline  reaction.  It  is  so 
sensitive  to  the  action  of  acids  that  at  22°  C.  its  devel- 
opment is  arrested  when  an  acid  reaction  equivalent  to 
0.066  to  0.08  per  cent,  of  hydrochloric  or  nitric  acid  is 
present.  (Kitasato.) 

Under  artificial  cultivation  the  maximum  develop- 
ment of  this  organism  is  reached  in  a  comparatively 
short  time ;  after  this  it  remains  quiescent  for  a  period, 
and  finally  degeneration  begins.  The  dying  comma 
bacilli  become  altered  in  appearance  and  assume  the 
condition  known  as  "  involution-forms."  (See  Fig.  74.) 
When  in  this  state  they  take  up  coloring-reagents  very 
faintly  or  not  at  all,  and  may  lose  entirely  their  charac- 
teristic shape. 

When  present  with  other  bacteria,  under  conditions 
favorable  to  growth,  the  comma  bacillus  at  first  grows 
much  more  rapidly  than  do  the  others ;  in  twenty-four 
hours  it  will  often  so  outnumber  the  other  organisms 
present  that  microscopic  examination  might  lead  one 
to  regard  the  material  under  consideration  as  a  pure 
culture  of  this  organism.  Its  conspicuous  develop- 
ment under  these  circumstances  does  not,  however,  last 
longer  than  two  or  three  days ;  degeneration  and  death 
begin,  and  the  other  organisms  gain  the  ascendency. 
This  fact  has  been  taken  advantage  of  by  Schottelius l 
in  the  following  method  devised  by  him  for  the  bac- 
teriological examination  of  dejections  from  cholera 
patients : 

In  dejections  not  examined  immediately  after  being 
passed  it  is  often  difficult,  because  of  the  large  num- 
ber of  other  bacteria  that  may  be  present,  to  detect 

1  Deutsche  med.  Wochenschrift,  1885,  No.  14. 


SPIRILLUM  CHOLERA  ASIATICS.  411 

with  certainty  the  cholera  organism  by  microscopic 
examination.  It  is  advantageous  in  these  cases  to  mix 
the  dejections  with  about  double  their  volume  of  slightly 
alkaline  beef-tea,  and  allow  them  to  stand  for  about 
twelve  hours  at  a  temperature  between  30°  and  40°  C. 
There  appears  at  the  end  of  this  time,  especially  upon 
the  surface  of  the  fluid,  a  conspicuous  increase  in  the 
number  of  comma  bacilli,  and  cover-slip  preparations 
made  from  the-  upper  layers  of  the  fluid  will  reveal  an 
almost  pure  culture  of  this  organism. 

It  is  not  improbable  that  a  similar  process  occurs  in 
the  intestines  of  those  suffering  from  Asiatic  cholera, 
viz.,  a  rapid  multiplication  of  the  comma  bacilli  that 
have  gained  access  to  the  intestines  takes  place,  but  lasts 
for  only  a  short  time,  when  the  comma  bacilli  begin  to 
disappear,  and  after  a  few  days  their  place  is  taken  by 
other  organisms. 

In  connection  with  his  experiments  upon  the  poison 
produced  by  the  cholera  organism  Pfeiffer1  states  that 
in  very  young  cultures,  grown  under  access  of  oxy- 
gen, there  is  present  a  body  that  possesses  intensely 
toxic  properties.  This  primary  cholera-poison  stands 
in  very  close  relation  to  the  material  composing  the 
bodies  of  the  bacteria  themselves,  and  is  probably  an 
integral  constituent  of  them,  for  the  vitality  of  the 
cholera  spirilla  can  be  destroyed  by  means  of  chloro- 
form or  thymol,  and  by  drying,  without  apparently 
any  alteration  of  this  poisonous  body.  Absolute  alco- 
hol, concentrated  solutions  of  neutral  salts,  and  a  tem- 
perature of  100°  C.,  decompose  this  substance,  leaving 
intact  secondary  poisons  which  possess  a  similar  physi- 
ological activity,  but  only  when  given  in  from  ten  to 
1  Zeitschrift  fur  Hygiene  und  Infektionskrankheiten,  Bd.  xi.  S.  393. 


412  BACTERIOLOGY. 

twenty  times  the  close  necessary  to  produce  the  same 
effects  with  the  primary  poison. 

Other  members  of  the  vibrio  family  also,  namely, 
vibrio  Metchnikovi  and  that  of  Finkler  and  Prior  (see 
description  of  these  species),  contain,  according  to  Pfeif- 
fer,  closely  related  poisons. 

EXPERIMENTS  UPON  ANIMALS.— As  a  result  of  ex- 
periments for  the  purpose  of  determining  if  the  disease 
can  be  produced  in  any  of  the  lower  animals  it  has  been 
found  that  white  mice,  monkeys,  cats,  dogs,  poultry,  and 
many  other  animals  are  not  susceptible  to  infection  by  the 
methods  usually  employed  in  inoculation  experiments. 
When  animals  are  fed  on  pure  cultures  of  the  comma 
bacillus  no  effect  is  produced,  and  the  organisms  cannot 
be  obtained  from  the  stomach  or  intestines.  They  are 
destroyed  in  the  stomach,  and  do  not  reach  the  intes- 
tines ;  they  are  not  demonstrable  in  the  faeces  of  these 
animals.  Intravascular  injections  of  a  pure  culture 
into  rabbits  are  followed  by  an  illness,  from  which 
the  animals  usually  recover  in  from  two  to  three  days ; 
intraperitoneal  injections  into  white  mice  are,  as  a  rule, 
followed  by  death  in  from  twenty-four  to  forty-eight 
hours ;  the  conditions  in  both  instances  most  probably 
resulting  from  the  toxic  activities  of  the  poisonous  prod- 
ucts of  growth  of  the  organisms  present  in  the  culture 
employed.  None  of  the  lower  animals  has  ever  been 
known  to  suffer  spontaneously  from  Asiatic  cholera. 

The  failure  to  induce  cholera  in  animals  by  feeding, 
or  by  injection  of  cultures  into  the  stomach,  was  shown 
by  Nicati  and  Kietsch1  to  be  due  to  the  destructive 
action  of  the  acid  gastric  juice  on  the  bacilli.  They 

1  Archiv.  de  Phys.  norm,  et  path.,  1885,  t.  vi.,  3e  ser.    Comptes  rendus, 
xcix.  p.  928.     Eevue  de  Hygiene,  1885.    Eevue  de  Medecine,  1885,  v. 


SPIRILLUM  CHOLERA  ASIATICS.  413 

showed  that  if  cultures  of  this  organism  were  intro- 
duced into  the  alimentary  tract  of  certain  animals  in 
such  a  manner  that  they  would  not  be  subjected  to  the 
influence  of  the  gastric  juice,  a  condition  pathologically 
closely  simulating  cholera  as  it  occurs  in  man  could  be 
produced.  For  this  purpose  the  common  bile-duct  was 
ligated,  after  which  the  cultures  were  injected  directly 
into  the  duodenum.  Such  interference  with  the  flow 
of  bile  lessens  intestinal  peristalsis,  and  thus  permits 
development  of  the  bacilli  at  the  point  at  which  they 
are  deposited — that  is,  the  portion  of  the  intestine  hav- 
ing an  alkaline  reaction  and  beyond  the  influence  of  the 
acid  stomach-juice. 

By  this  method  Nicati  and  Rietsch,  Van  Ermengem,1 
Koch,2  and  others  were  enabled  to  produce  in  the  ani- 
mals upon  which  they  operated  a  condition  that  was,  if 
not  identical,  at  all  events  very  similar  pathologically  to 
that  seen  in  the  intestines  of  subjects  dead  of  the  disease. 

At  a  subsequent  conference  held  in  Berlin  in  1885 
Koch3  described  the  following  method,  by  means  of 
which  he  had  been  able  to  obtain  a  much  greater  de- 
gree of  constancy  in  all  his  efforts  to  produce  cholera  in 
lower  animals:  bearing  in  mind  the  point  made  by 
Nicati  and  Rietsch  as  to  the  effect  produced  by  the  acid 
reaction  of  the  gastric  juice,  this  reaction  was  first  to  be 
neutralized  by  injecting  through  a  soft  catheter  passed 
down  the  oesophagus  into  the  stomach  5  c.c.  of  a  5  per 
cent,  solution  of  sodium  carbonate.  Ten  or  fifteen  min- 
utes later  this  was  to  be  followed  by  the  injection  into 
the  stomach  (also  through  a  soft  catheter)  of  10  c.c.  of  a 

1  Recherches  sur  le  Microbe  du  Cholera  Asiatique,  Paris-Bruxelles, 
1885.     Bull,  de  1'Acad.  roy.  de  Med.  de  Belgique,  xviii.,  3e  ser. 

2  Loc.  cit.  »  Ibid.,  1885. 


414  BACTERIOLOGY. 

bouillon  culture  of  the  cholera  spirillum.  For  the  pur- 
pose of  arresting  peristalsis  and  permitting  the  bac- 
teria to  remain  in  the  stomach  and  upper  part  of  the 
duodenum  for  as  long  a  time  as  possible,  the  animal  was 
to  receive,  immediately  following  the  injection  of  the 
culture,  an  intraperitoneal  injection,  by  means  of  a 
hypodermic  syringe,  of  1  c.c.  of  tincture  of  opium  for 
each  200  grammes  of  its  body-weight.  Shortly  after 
this  last  injection  deep  narcosis  sets  in  and  lasts  from 
a  half  to  one  hour,  after  which  the  animal  is  as  lively 
as  ever.  Of  35  guinea-pigs  inoculated  in  this  way  by 
Koch,  30  died  of  an  affection  that  was,  in  general,  very 
similar  to  Asiatic  cholera  as  seen  in  man. 

The  condition  of  those  animals  before  death  is  de- 
scribed as  follows :  twenty-four  hours  after  the  opera- 
tion the  animal  appears  unwell ;  there  is  loss  of  appetite, 
and  the  animal  remains  quiet  in  its  cage.  On  the  fol- 
lowing day  a  paralytic  condition  of  the  hind  extremities 
appears,  which,  as  the  day  wears  on,  becomes  more 
pronounced  ;  the  animal  lies  quite  flat  upon  its  abdomen 
or  on  its  side,  with  legs  extended ;  respiration  is  weak 
and  prolonged,  and  the  pulsations  of  the  heart  are  hardly 
perceptible  ;  the  head  and  extremities  are  cold,  and  the 
body-temperature  is  frequently  subnormal.  The  ani- 
mal usually  dies  after  remaining  in  this  condition  for  a 
few  hours. 

At  autopsy  the  small  intestine  is  found  deeply  in- 
jected and  filled  with  flocculent,  colorless  fluid.  The 
stomach  and  intestines  do  not  contain  solid  masses,  but 
fluid ;  when  diarrhoea  does  not  occur,  firm  scybala  may 
be  detected  in  the  rectum.  Both  by  microscopic  exam- 
ination and  by  culture  methods  comma  bacilli  are  found 
present  in  the  small  intestine  in  practically  pure  culture. 


SPIRILLUM  CHOLERA  ASIATICS.  415 

More  recently  Pfeiffer1  has  determined  that  essen- 
tially similar  constitutional  effects  may  be  produced  in 
guinea-pigs  by  the  intraperitoneal  injection  of  rela- 
tively large  numbers  of  this  organism.  His  plan  is  to 
scrape  from  the  surface  of  a  fresh  culture  on  agar-agar 
as  much  of  the  growth  as  can  be  held  upon  a  medium 
size  wire  loop.  This  is  then  finely  divided  in  1  c.c. 
of  bouillon,  and  by  means  of  a  hypodermic  syringe  is 
injected  directly  "into  the  peritoneal  cavity.  When  vir- 
ulent cultures  have  been  used  this  operation  is  quickly 
followed  by  a  fall  in  the  temperature  of  the  animal  that 
is  gradual  and  continuous  until  death  ensues,  which  usu- 
ally occurs  in  from  eighteen  to  twenty-four  hours  after 
the  operation,  though  exceptionally  the  animal  recovers, 
even  after  having  exhibited  marked  symptoms  of  pro- 
found toxaemia. 

Continuing  his  studies  upon  this  disease,  Pfeiffer 2  has 
demonstrated  that  it  is  possible  to  render  an  animal  tol- 
erant to  or  immune  from  the  poisonous  properties  of  this 
organism  by  repeated  injections  of  non-fatal  doses  of 
dead  cultures  (cultures  that  have  been  killed  by  the 
vapor  of  chloroform  or  by  heat).  He  also  demon- 
strated that  animals  so  immunized  possess  a  specific 
^  rmicidal  action  toward  the  cholera  spirillum — i.  e.,  if 
into  the  peritoneal  cavity  of  an  animal  immunized  from 
Asiatic  cholera  living  cholera  spirilla  be  introduced, 
they  will  all  be  destroyed  (disintegrated)  within  a  rela- 
tively short  time.  Furthermore,  if  the  serum  of  an  animal 
immunized  from  cholera  be  injected  into  the  peritoneal 
cavity  of  another  animal  of  the  same  species,  but  not  so 

1  Zeitschrift  fur  Hygiene  und  Infektionskrankheiten,  Bd.  xi.  and  xiv. 

2  Ibid.,  1894,  Bd.  xvii.  S.  355;  1894,  Bd.  xviii.  S.  1 ;  1895,  Bd.  xx. 
S.  197. 


416  BACTERIOLOGY. 

protected,  and  immediately  afterward  living  cholera  spir- 
illa be  introduced,  a  similar  disintegration  and  destruction 
of  the  bacteria  will  also  result.  He  shows  that  a  more  or 
less  definite  relation  exists  between  the  amount  of  serum 
and  the  number  of  organisms  introduced.  Such  a  de- 
struction of  the  comma  bacillus  by  the  serum  of  an 
immunized  animal  does  not  occur  outside  the  animal 
body — that  is,  it  cannot  be  demonstrated  in  a  test-tube. 
The  specificity  of  this  reaction  is  suggested  by  Pfeiffer 
as  a  means  of  differentiating  the  cholera  spirillum  from 
other  suspicious  species,  for  no  such  bacteriolytic  action 
is  observed  if  species  other  than  the  cholera  spirillum 
be  introduced  into  the  peritoneal  cavity  of  animals  im- 
munized from  Asiatic  cholera. 

Pfeiffer  has  further  demonstrated  that  the  serum  of 
animals  artificially  immunized  from  Asiatic  cholera  has 
an  agglutinating  effect  upon  fluid  cultures  of  the  cholera 
spirillum  similar  to  that  seen  when  typhoid  bacilli  are 
mixed  with  serum  from  typhoid  cases,  or  from  animals 
artificially  immunized  from  typhoid  infection  or  intoxi- 
cation. (See  Agglutinin.) 

GENERAL  CONSIDERATIONS. — In  all  cases  of  Asiatic 
cholera,  and  only  in  this  disease,  the  organism  just 
described  can  be  detected  in  the  intestinal  evacuations. 
The  more  acute  the  case  and  the  more  promptly  the 
examination  is  made  after  the  evacuations  have  passed 
from  the  patient,  the  less  difficulty  will  be  experienced 
in  detecting  the  organism. 

In  some  cases  the  organism  can  be  detected  in  the 
vomited  matters,  though  by  no  means  so  constantly  as 
in  the  intestinal  contents. 

As  a  rule,  bacteriological  examination  fails  to  reveal 
the  presence  of  the  organisms  in  the  blood  and  internal 


SPIRILLUM  CHOLERA  ASIATICS.  417 

in  this  disease,  though  Nicati  and  Rietsch  claim 
to  have  obtained  them  from  the  common  bile-duct  in 
rapidly  fatal  cases,  and  in  two  out  of  five  cases  they 
were  present  in  the  gall-bladder.  Doyen  and  Rasst- 
sehewsky l  found  them  in  the  liver  in  pure  culture,  and 
Tizzoni  and  Cattani2  in  both  the  blood  and  the  gall- 
bladder. 

The  cholera  spirillum  is  a  facultative  parasite ;  that 
is  to  say,  it  apparently  finds  in  certain  parts  of  the 
world,  particularly  in  those  countries  in  which  Asiatic 
cholera  is  endemic,  conditions  that  are  not  entirely  un- 
favorable to  its  development  outside  of  the  body.  This 
has  been  found  to  be  the  case  not  only  by  Koch,  who 
detected  the  presence  of  the  organism  in  water-tanks 
in  India,  but  by  many  other  observers  who  have  suc- 
ceeded in  demonstrating  its  growth  under  conditions  not 
embraced  in  the  ordinary  methods  employed  for  the 
cultivation  of  bacteria.3 

The  results  of  experiments  having  for  their  object 
the  determination  of  the  length  of  time  during  which 
this  organism  may  retain  its  vitality  in  water  are  con- 
spicuous for  their  irregularity.  In  the  transactions  of 
the  congress  in  Berlin  for  the  discussion  of  the  cholera 
question,  it  is  stated,  in  connection  with  this  point,  that 
the  experiments  made  writh  tank-water  in  India  some- 
times resulted  in  demonstrating  the  multiplication  of 
the  organisms  introduced  into  it,  while  in  other  cases 
they  died  very  quickly. 

1  Reference  to  Vratch,  1885,  in  Allg.  Med.  Central  Zeitung,  Berlin. 

2  Centralblatt  fur  die  med.  Wissenschafteu,  1886,  Xo.  43. 

3  Obviously  all  pathogenic  bacteria  that  have  been  isolated  under 
artificial  methods  of  cultivation  are  facultative  parasites.    Were  they 
obligate  parasites  their    cultivation    upon  dead  materials  would    be 
impossible. 

27 


418  BACTERIOLOGY. 

On  February  8,  1884,  comma  bacilli  were  found  in  a 
tank  at  Saheb-Began,  in  Calcutta,  and  it  was  possible 
to  demonstrate  them  in  a  living  condition  up  to  Feb- 
ruary 23d. 

Koch  states  that  in  ordinary  spring-water  or  well- 
water  the  organisms  retained  their  vitality  for  thirty 
days,  whereas  in  the  canal-water  (sewage)  of  Berlin  they 
died  after  six  or  seven  days ;  but  if  this  latter  were 
mixed  with  faecal  matters,  the  organisms  retained  their 
vitality  for  but  twenty-seven  hours ;  and  in  the  undi- 
luted contents  of  cesspools  it  was  impossible  to  demon- 
strate them  after  twelve  hours.  In  the  experiments  of 
Nicati  and  Rietsch  they  retained  their  vitality  in  steril- 
ized distilled  water  for  twenty  days ;  in  Marseilles  canal- 
water  (sewage),  for  thirty-eight  days ;  in  sea-water, 
sixty-four  days ;  in  harbor-water,  eighty-one  days ;  and 
in  bilge-water,  thirty-two  days. 

In  the  experiments  of  Hochstetter,  on  the  other  hand, 
they  died  in  distilled  water  in  less  than  twenty-four 
hours  in  five  of  seven  experiments ;  in  one  of  the  two 
remaining  experiments  they  were  alive  after  a  day,  and 
in  the  other  after  seven  days. 

In  one  test  with  the  water-supply  of  Berlin  the 
organism  retained  its  vitality  for  267  days,  and  in 
another  for  382  days,  notwithstanding  the  fact  that 
many  other  organisms  were  present  at  the  same  time. 
There  is  no  ready  explanation  for  these  variations, 
for  they  depend  apparently  upon  a  number  of  factors 
which  may  act  singly  or  together.  For  example,  in 
general  it  may  be  said  that  the  higher  the  tempera- 
ture of  the  water  in  which  these  organisms  are  present, 
up  to  20°  C.,  the  longer  do  they  retain  their  vitality ; 
the  purer  the  water — that  is;  the  poorer  in  organic 


SPIRILLUM  CHOLERA  ASIATICS.  419 

matters — the  more  quickly  do  the  organisms  die,  whereas 
the  richer  it  is  in  organic  matter  the  longer  do  they 
retain  their  vitality. 

Still  another  point  that  must  be  considered  in  this 
connection  is  the  antagonistic  influences  under  which 
they  find  themselves  when  placed  in  water  containing 
large  numbers  of  organisms  that  are,  so  to  speak,  at 
home  in  water — the  so-called  normal  water-saprophytes. 

The  effect  of  light  upon  growing  bacteria  must  not 
be  lost  sight  of,  for  it  has  been  shown  that  a  surprisingly 
large  number  of  these  organisms  are  robbed  of  their 
vitality  by  a  relatively  short  exposure  to  the  rays  of 
the  sun ;  and  it  is  therefore  not  unlikely  that  the  non- 
observance  of  this  fact  may  be,  in  part  at  least,  account- 
able for  some  of  the  discrepancies  that  appear  in  the 
results  of  these  experiments. 

In  his  studies  upon  the  behavior  of  pathogenic  and 
other  micro-organisms  in  the  soil  Carl  Frankel l  found 
that  the  cholera  spirillum  was  not  markedly  susceptible 
to  those  deleterious  influences  that  cause  the  death  of 
a  number  of  other  pathogenic  organisms.  During  the 
months  of  August,  September,  and  October  cultures  of 
the  comma  bacillus  that  had  been  buried  in  the  ground 
at  a  depth  of  three  metres  retained  their  vitality ;  on 
the  other  hand,  in  other  months,  particularly  from 
April  to  July,  they  lost  their  vitality  when  buried  to 
the  depth  of  only  two  metres.  At  a  depth  of  one  and 
a  half  metres  vitality  was  not  destroyed,  and  there  was 
a  regular  development  in  cultures  so  placed. 

As  a  result  of  experiments  performed  in  the  Imperial 
Health  Bureau  at  Berlin,  it  was  found  that  the  bodies 
of  guinea-pigs  that  had  died  of  cholera  induced  by 

1  Zeitschrift  fur  Hygiene,  Bd.  ii.  8.  521. 


420  BACTERIOLOGY. 

Koch's  method  of  inoculation  contained  no  living 
cholera  spirilla  when  exhumed  after  having  been 
buried  for  nineteen  days  in  wooden  boxes,  or  for 
twelve  days  in  zinc  boxes.  In  a  few  that  had  been 
buried  in  moist  earth,  without  having  been  encased  in 
boxes,  when  exhumed  after  two  or  three  months,  the 
results  of  examinations  for  cholera  spirilla  were  like- 
wise negative. 

Kitasato,1  in  his  experiments  with  the  cholera  organ- 
ism, found  that  when  mixed  with  the  intestinal  evacu- 
ations of  human  beings  under  ordinary  conditions  it 
lost  its  vitality  in  from  a  day  and  a  half  to  three 
days.  If  the  evacuations  were  sterilized  before  the 
cultures  were  mixed  with  them,  the  organisms  retained 
their  vitality  from  twenty  to  twenty-five  days.  He 
was  unable  to  come  to  any  definite  conclusion  as  to  the 
cause  of  these  phenomena. 

It  was  demonstrated  by  Hesse 2  and  by  Celli 3  that 
many  substances  commonly  employed  as  food-stuffs 
serve  as  favorable  materials  for  the  development  of 
the  cholera  organisms.  In  his  experiments  upon  its 
behavior  in  milk  Kitasato 4  found  that  at  a  temperature 
of  36°  C.  the  cholera  spirillum  developed  very  rapidly 
during  the  first  three  or  four  hours,  and  outnumbered 
the  other  organisms  commonly  found  in  milk.  They 
then  diminished  in  number  from  hour  to  hour  as  the 
acidity  of  the  milk  increased,  until  finally  their  vitality 
was  lost ;  at  the  same  time  the  common  saprophytic 
bacteria  increased  in  number.  Kelatively  the  same 
process  occurs  at  a  lower  temperature,  from  22°  to  25° 

1  Zeitschrift  fur  Hygiene,  Bd.  v.  S.  487. 

2  Ibid.,  Bd.  v..S.  527. 

3  Bolletino  della  E.  Acad.  Med.  di  Roma,  1888. 

4  Zeitschrift  fur  Hygiene,  Bd.  v.  S.  491. 


SPIRILLUM  CHOLERA  ASIATICS.  421 

C. ;  but  it  is  slower,  the  maximum  development  of  the 
cholera  organisms  being  reached  at  about  the  fifteenth 
hour,  after  which  time  they  were  outnumbered  by  the 
ordinary  saprophytes  present. 

From  the  foregoing  it  would  seem  that  the  vitality  of 
the  cholera  spirillum  in  milk  depends  largely  upon  the 
reaction  :  the  more  quickly  the  milk  becomes  sour  the 
more  quickly  does  the  organism  become  inert;  while 
the  longer  the  milk  retains  its  neutral,  or  only  very 
slightly  acid,  reaction,  the  longer  do  the  cholera  organ- 
isms that  may  be  present  in  it  retain  their  power  of 
multiplication. 

According  to  Laser/  the  cholera  organism  retains  its 
vitality  in  butter  for  about  seven  days ;  it  is  therefore 
possible  for  the  disease  to  be  contracted  by  the  use  of 
butter  that  has  in  any  \vay  been  in  contact  with  cholera 
material. 

In  regard  to  the  antagonism  between  the  cholera 
spirillum  and  other  organisms  with  which  it  may  come 
in  contact,  the  experiments  of  Kitasato2  led  him  to 
conclude  that  no  organism  has  been  found  which, 
when  growing  in  the  same  culture-medium  with  it, 
possessed  the  power  of  depriving  it  of  vitality  within 
a  short  time.  On  the  other  hand,  the  experiments 
showed  that  there  were  quite  a  number  of  other  organ- 
isms the  development  of  which  was  checked,  and  in 
some  cases  their  vitality  was  completely  destroyed, 
when  growing  in  the  same  medium  with  the  cholera 
spirillum. 

From  this  it  would  appear  that  the  disappearance  of 
the  cholera  spirillum  from  mixed  cultures  and  from  the 
evacuations  in  the  short  time  mentioned,  is  due  more 

1  Zeitschrift  fur  Hygiene.  Bd.  x.  S.  513.  2  Ibid.,  Bd.  vi.  S.  1. 


422  BACTERIOLOGY. 

to  unfavorable  nutritive  conditions  than  to  the  direct 
action  of  the  other  organisms  present. 

When  completely  dried,  according  to  Koch,  the 
cholera  spirillum  does  not  retain  its  vitality  longer 
than  twenty-four  hours,  but  by  others  its  vitality  is 
said  to  be  destroyed  by  an  absolute  drying  of  three 
hours.  In  moist  conditions,  as  in  artificial  cultures, 
vitality  may  be  retained  for  many  months ;  though  re- 
peated observations  lead  us  to  believe  that  under  these 
circumstances  virulence  is  diminished.  According  to 
Kitasato,1  they  retain  their  vitality  when  smeared  upon 
thin  glass  cover-slips  and  kept  in  the  moist  chamber 
for  from  85  to  100  days,  and  for  as  long  as  200  days 
when  deposited  upon  bits  of  silk  thread. 

In  the  course  of  his  studies  upon  the  persistency  of 
pathogenic  micro-organisms  in  the  dead  body  von 
Esmarch 2  found  that  when  the  cadaver  of  a  guinea- 
pig  dead  after  the  introduction  of  cholera  organisms 
into  the  stomach  was  immersed  in  water  until  decom- 
position set  in,  after  eleven  days,  when  decomposi- 
tion was  far  advanced,  it  was  impossible  to  find  any 
living  cholera  spirilla  by  the  ordinary  plate  methods. 

A  similar  experiment  resulted  in  their  disappearance 
in  five  days.  In  another  experiment,  in  which  de- 
composition was  allowed  to  go  on  without  the  animal 
being  immersed  in  water,  none  could  be  detected  after 
the  fifth  day. 

Carl  Frankel 3  has  shown  that  an  atmosphere  of  car- 
bonic acid  is  directly  inhibitory  to  the  development  of 
the  cholera  spirillum,  and  Percy  Frankland 4  states  that 
in  an  atmosphere  of  this  gas  it  dies  in  about  eight  days. 

1  Zeitschrift  fur  Hygiene,  Bd.  v.  S.  134.  2  Ibid.,  Bd.  vii.  S.  1. 

3  Ibid.,  Bd.  v.  S.  332.  *  Ibid.,  Bd.  vi.  S.  13. 


THE  DIAGNOSIS  OF  ASIATIC  CHOLERA.      423 

In  an  atmosphere  of  carbon  monoxide  its  vitality  is  lost~ 
in  nine  days,  and  in  general  the  same  may  be  said  for  it 
when  exposed  to  an  atmosphere  of  nitrous  oxide  gas. 

From  what  has  been  said,  we  see  that  the  spirillum 
of  Asiatic  cholera,  while  possessing  the  power  of  pro- 
ducing in  human  beings  one  of  the  most  rapidly  fatal 
diseases  with  which  we  are  acquainted,  is  still  one  of  the 
least  resistant  of  the  pathogenic  organisms  known  to  us. 
Under  conditions  most  favorable  to  its  growth  its  de- 
velopment is  self-limited ;  it  is  markedly  susceptible  to 
acids,  alkalies,  other  chemical  disinfectants,  and  heat ; 
but  when  partly  dried  upon  clothing,  food,  or  other 
objects,  it  may  retain  its  vitality  for  a  relatively  long 
period  of  time,  and  it  is  more  than  probable  that  in  this 
way  the  disease  is  often  disseminated  from  points  in 
which  it  is  epidemic  or  endemic  into  localities  that  are 
free  from  it. 

THE    DIAGNOSIS    OF   ASIATIC    CHOLERA   BY   BACTERIO- 
LOGICAL  METHODS. 

Because  of  the  manifold  channels  that  are  open  for 
the  dissemination  of  this  disease  it  is  of  the  utmost 
importance  that  it  should  be  recognized  as  quickly 
as  possible,  for  with  every  moment  of  delay  in  its 
recognition  opportunities  for  its  spread  multiply.  It 
is  essential,  therefore,  when  employing  bacteriological 
means  for  making  the  diagnosis,  to  bear  in  mind  those 
biological  and  morphological  features  of  the  organism 
that  appear  most  quickly  under  artificial  methods  of 
cultivation,  and  which,  at  the  same  time,  may  be  con- 
sidered as  characteristic  of  it,  viz.,  its  peculiar  mor- 
phology and  grouping ;  the  much  greater  rapidity  of  its 
growth  over  that  of  other  bacteria  with  which  it  may 


424  BACTERIOLOGY. 

be  associated ;  the  characteristic  appearance  of  its  col- 
onies on  gelatin  plates  and  of  its  growth  in  stab-cultures 
in  gelatin ;  its  property  of  producing  indol  and  coinci- 
dently  nitrites  in  from  six  to  eight  hours  in  peptone 
solution  at  37°  to  38°  C. ;  and  its  power  of  causing  the 
death  of  guinea-pigs  in  from  sixteen  to  twenty-four 
hours  when  introduced  into  the  peritoneal  cavity,  death 
being  preceded  by  symptoms  of  extreme  toxsemia,  char- 
acterized by  prostration  and  gradual  and  continuous 
fall  in  the  temperature  of  the  animal's  body. 

In  1893  Koch1  called  attention  to  a  plan  of  pro- 
cedure that  comprehends  the  points  just  enumerated. 
By  its  employment  the  diagnosis  can  be  established  in 
the  majority  of  cases  of  Asiatic  cholera  in  from  eighteen 
to  twenty-two  hours.  In  general,  the  steps  to  be  taken 
and  points  to  be  borne  in  mind  are  as  follows :  the 
evacuations  should  be  examined  as  soon  as  possible 
after  they  have  been  passed. 

MICROSCOPIC  EXAMINATION. — 1.  From  one  of  the 
small  slimy  particles  seen  in  the  semi-fluid  evacuations 
prepare  a  cover-slip  preparation  in  the  ordinary  way 
and  stain  it.  If,  upon  microscopic  examination,  only 
curved  rods,  or  curved  rods  greatly  in  excess  of  all 
other  forms,  are  present,  the  diagnosis  of  Asiatic  cholera 
is  more  than  likely  correct ;  and  particularly  is  this  true 
if  these  organisms  are  arranged  in  irregular  linear 
groups  with  the  long  axes  of  all  the  rods  pointing  in 
nearly  the  same  direction — that  is  to  say,  somewhat  as 
minnows  arrange  themselves  when  swimming  up  stream 
in  schools.  (Koch.) 

In  1886  Weisser  and  Frank2  expressed  their  opinion 

1  Zeitschrift  fur  Hygiene  und  Infiktionskleiten,  1893,  Bd.  xiv.  S.  319. 

2  Ibid.,  1886,  Bd.  i.  S.  379. 


THE  DIAGNOSIS  OF  ASIATIC  CHOLERA.      425 

upon  the  value  of  microscopic  examination  in  these  a 
as  follows : 

a.  In  the  majority  of  cases  microscopic  examination 
is  sufficient  for  the  detection  of  the  presence  of  the 
comma  bacillus  in  the  intestinal  evacuations  of  cholera 
patients. 

6.  Even  in  the  most  acute  cases,  running  a  very  rapid 
course,  the  comma  bacillus  can  always  be  found  in  the 
evacuations. 

c.  In  general,  the  number  of  cholera  spirilla  present 
is  greater  the  earlier  death  occurs ;  when  death  is  de- 
layed, and   the  disease   continues   for  a   long   period, 
their  number  is  diminished. 

d.  Should  the  patient  not  die  of  cholera,  but  of  some 
other  disease,  such  as  typhoid  fever,  that  may  be  en- 
grafted upon  it,  comma  bacilli  may  disappear  entirely 
from  the  intestines. 

2.  From  another  slimy  flake  prepare  a  set  of  gelatin 
plates.     Expose  them  to  a  temperature  of  from  20°  to 
22°  C.,  and  after  sixteen,  twenty-two,  and   thirty-six 
hours  observe  the  appearance  of  the  colonies.     Usually 
after  about  twenty-two  hours  the  colonies  of  this  organ- 
ism can  easily  be  identified  by  one  familiar  with  them. 

3.  With  another  slimy  flake  start  a  culture  in  a  tube 
of  peptone  solution — either  the   solution   of   Dunham 
or,  as  Koch  proposes,  a  solution  of  double  the  strength 
of  that  of  Dunham  (Witte's  peptone  is  to  be  used,  as  it 
gives  the  best  and  most  constant  results).     Keep  this  at 
from  37°  to  38°  C.,  and  at  the  end  of  from  six  to  eight 
hours  prepare  cover-slips  from  the  upper  layers  (without 
si  la  king)  and  examine  them  microscopically.    If  comma 
bacilli  were  present  in  the  original  material,  and  are 
capable  of  multiplication,  they  will  be  found  in  this  local- 


426  BACTERIOLOGY. 

ity  in  almost  pure  culture.  After  the  microscopic  exami- 
nation prepare  a  second  peptone  culture  from  the  upper 
layers  of  the  one  just  examined,  also  a  set  of  gelatin 
plates,  and  with  what  remains  make  the  test  for  indol 
by  iLe  addition  of  10  drops  of  concentrated  sulphuric 
acid  for  each  10  c.c.  of  fluid  contained  in  the  tube.  If 
comma  bacilli  are  growing  in  the  tube,  the  rose  color 
characteristic  of  the  presence  of  indol  should  appear. 

By  following  this  plan  "a  bacteriologist  who  is 
familiar  with  the  morphological  and  biological  peculi- 
arities of  this  organism  should  make  a  more  than  prob- 
able diagnosis  at  once  by  microscopic  examination  alone, 
and  a  positive  diagnosis  in  from  twenty  to,  at  most, 
twenty-four  hours  after  beginning  the  examination/7 
(Koch.) 

In  certain  doubtful  cases  the  organisms  are  present  in 
the  intestinal  canal  in  very  small  numbers,  and  micro- 
scopic examination  is  not,  therefore,  of  so  much  assist- 
ance. In  these  cases  plates  of  agar-agar,  of  gelatin, 
and  cultures  in  the  peptone  solution  should  be  made. 

The  plates  of  agar-agar  should  not  be  prepared  in 
the  usual  way,  but  the  agar-agar  should  be  poured  into 
Petri  dishes  and  allowed  to  solidify,  after  which  one  of 
the  slimy  particles  may  be  smeared  over  its  surface. 
The  comma  bacillus,  being  markedly  aerobic,  develops 
very  much  more  readily  when  its  colonies  are  located 
upon  the  surface  then  when  in  the  depths  of  the  med- 
ium. A  point  to  which  Koch  calls  attention,  in  con- 
nection with  this  step  in  manipulation,  is  the  necessity 
of  having  the  surface  of  the  agar-agar  free  from 
the  water  squeezed  from  it  when  it  solidifies,  as  the 
presence  of  the  water  interferes  with  the  development 
of  the  colonies  at  isolated  points  and  causes  them  to 


THE  DIAGNOSIS  OF  ASIATIC  CHOLERA.      427 

become  confluent.  To  obviate  this  he  recommends  that 
the  agar-agar  be  poured  into  the  plates  and  the  water 
allowed  to  separate  from  the  surface  at  the  temperature 
of  the  incubator  before  they  are  used.  It  is  wise,  there- 
fore, when  one  is  liable  to  be  called  on  for  such  work 
as  this,  to  keep  a  number  of  sterilized  plates  of  agar- 
agar  in  the  incubator  ready  for  use,  just  as  sterilized 
tithe*  of  the  media  are  always  ready  at  hand.  The 
advantage  of  using  the  agar  plates  is  the  higher  tem- 
perature at  which  they  can  be  kept,  and  consequently 
a  more  favorable  condition  for  the  development  of  the 
colonies. 

As  soon  as  isolated  colonies  appear  they  should 
be  examined  microscopically  for  the  presence  of  bac- 
teria having  the  morphology  of  the  one  we  are  seek- 
ing, and  as  soon  as  such  is  detected  gelatin  plates 
and  cultures  in  peptone  solution  (for  the  indol  reac- 
tion) should  be  made.  The  peptone  culture  from  the 
original  material  should  be  examined  microscopically 
from  hour  to  hour  after  the  sixth  hour  that  it  has 
been  in  the  incubator.  The  material  taken  for  ex- 
amination should  always  come  from  near  the  surface 
of  the  fluid,  and  care  should  be  taken  not  to  shake 
the  tube.  As  soon  as  comma  bacilli  are  detected  in 
considerable  numbers  in  the  upper  layers  of  the 
fluid  agar-agar  plates  and  fresh  peptone  cultures 
should  be  made  from  them.  In  from  ten  to  twelve 
hours  at  37°  C.  the  colonies  will  develop  on  the  agar- 
agar  plates  to  a  size  sufficient  for  recognition  by  micro- 
scopic examination,  and  from  this  examination  an 
opinion  can  usually  be  formed.  This  opinion  should 
always  be  controlled  by  cultures  in  the  peptone  solution 
made  from  each  of  several  single  colonies,  and  finally 


428  BACTERIOLOGY. 

the  test  for  the  presence  or  absence  of  indol  in  these 
cultures  should  be  made. 

In  all  doubtful  cases,  in  which  only  a  few  curved 
bacilli  are  present,  or  in  which  irregularities  in  either 
the  rate  or  mode  of  their  development  occur,  pure  cult- 
ures should  be  obtained  as  soon  as  possible  by  the  agar- 
agar  plate  method  and  by  the  method  of  cultivation  in 
peptone  solution,  and  their  virulence  tested  upon  ani- 
mals. For  this  purpose  cultures  upon  agar-agar  from 
single  colonies  must  be  made.  From  the  surface  of 
one  of  such  cultures  a  large  wire-loopful  should  be 
scraped  and  broken  up  in  about  one  cubic  centimetre 
of  bouillon,  and  the  suspension  thus  made  injected 
by  means  of  a  hypodermic  syringe  directly  into  the 
peritoneal  cavity  of  a  guinea-pig  of  about  350  to 
400  grammes  weight.  For  larger  animals  more  mate- 
rial is  used.  If  the  material  injected  is  from  a 
fresh  culture  of  the  cholera  organism,  toxic  symp- 
toms at  once  appear;  these  have  their  most  pro- 
nounced expression  in  depression  of  temperature,  and 
if  one  follows  this  decline  in  temperature  from  time  to 
time  with  the  thermometer  it  will  be  seen  to  be  gradual 
and  continuous  from  the  time  of  injection  to  the  death 
of  the  animal  (Pfeiffer1),  which  occurs  in  from  eighteen 
to  twenty-four  hours  after  the  operation. 

In  general,  this  is  the  procedure  employed  in  the  In- 
stitute for  Infectious  Diseases  at  Berlin,  under  Koch's 
direction. 

1  Loc.  cit. 


CHAPTER    XXIII. 

Spirilla  of  interest,  historically  and  otherwise,  that  have  been  con- 
founded with  the  spirillum  of  Asiatic  cholera — Their  peculiarities 
and  differential  features — Vibrio  proteus,  or  bacillus  of  Finkler  and 
Prior — Spirillum  tyrogenum,  or  cheese  spirillum  of  Deneke — The 
spirillum  of  Miller — Vibrio  Metchnikovi. 

VIBRIO   PROTEUS   (FINKLER-PRIOR   BACILLUS). 

FINKLER  AND  PRIOR  were  the  first  to  question  experi- 
mentally the  significance  of  the  presence  of  Koch's 
comma  bacillus  in  Asiatic  cholera,  claiming  to  have 
found  it  in  the  dejections  of  individuals  suffering  from 
other  maladies,  particularly  cholera  nostras.  The  mor- 
phological and  biological  differences  between  the  or- 
ganism that  Finkler  and  Prior  discovered  and  the 
comma  bacillus  described  by  Koch,  are,  however,  so 
pronounced  as  to  warrant  the  opinion  that  the  confusion 
arose  through  imperfect  and  untrustworthy  methods  of 
experimentation.  Subsequently  Finkler  and  Prior  re- 
tracted their  claims  of  identity  for  the  two  organisms, 
and  held  that  the  bacterium  with  which  they  were  deal- 
ing was  peculiar  to  cholera  nostras — an  opinion  which, 
in  the  light  of  later  work,  was  also  proved  to  be  with- 
out foundation. 

The  characteristics  of  the  spirillum  of  Finkler  and 
Prior  are  as  follows : 

MORPHOLOGY. — It  is  thicker  and  longer  than  the 
spirillum  of  Asiatic  cholera ;  it  is  often  thicker  at  the 
middle  than  at  the  poles ;  it  forms,  like  the  "  comma 
bacillus,"  screw-like,  twisted  threads.  (Fig.  77.) 

429     fr 


430  BACTERIOLOGY. 

It  is  supplied  with  a  single  flagellum  at  one  of  its 
ends,  and  is  therefore  motile. 


FIG.  77. 


*         (    '         X 

-^  -<    f^* 

~-  ,J  f 

Vibrio  proteus  (Finkler-Prior  bacillus),  from  culture  on  agar-agar  twenty- 
four  hours  old. 

It,  like  the  comma  bacillus,  readily  undergoes  degen- 
erative changes  under  conditions  unfavorable  to  growth, 
and  presents  the  variety  of  shapes  grouped  under  the 
head  "  involution-forms."  According  to  Buchner,  this 
is  especially  the  case  when  the  medium  in  which  they 
are  growing  contains  glucose  (5  per  cent.)  or  glycerin 
(2  per  cent.). 

CULTURAL  PECULIARITIES.  —  On  gelatin  plates  the 
development  of  its  colonies  is  far  more  rapid,  and  lique- 
faction far  more  extensive,  than  in  the  case  of  the 
cholera  spirillum.  After  twenty-two  to  twenty-four 
hours  in  this  medium  at  20°  to  22°  C.  the  average  size  of 
the  colonies  is  about  double  that  of  the  comma  bacillus. 
The  colonies  are  darker  and  denser,  and  do  not  pre- 
sent under  a  low-power  lens  the  same  degree  of  granula- 
tion and  subsequent  lobulation,  and  they  do  not  become 
serrated  or  scalloped  around  the  margin,  as  is  the  case 
with  Koch's  organism.  After  twenty-two  to  twenty- 
four  hours  they  are  usually  nearly  round,  regularly 
granular,  and  more  or  less  sharply  defined.  (See  Fig. 
78,  a.)  At  times  they  may  show  indefinite  markings 
or  creases,  somewhat  suggestive  of  lobulations.  After 


VIBRIO  PROTEUS.  431 

forty-eight  hours  on  gelatin  they  usually  range  from 
one  to  three  millimetres  (some  even  larger)  in  diameter, 
and  appear  as  sharply  cut,  saucer-shaped  pits  of  lique- 
faction, in  the  most  dependent  portion  of  which  lies 
a  dense,  irregular  mass,  the  colony  proper.  Under  a 
low  magnifying  power  they  present  at  this  stage  an  ap- 
pearance similar  to  that  shown  in  Fig.  78,  6,  the  central 
dense  mass  representing  the  colony,  and  the  irregular, 

FIG.  78. 


• 

Colonies  of  the  Finkler-Prior  bacillus  on  gelatin,    x  about  75  diameters. 

a.  After  twenty-two  hours  at  20°  to  22°  C.     &.  After  forty-eight  hours  at 

20°  to  22°  C. 

ragged  lines  surrounding  it  being  shreds  that  have  be- 
come torn  away  as  it  sank  into  the  liquid  caused  by  its 
growth.  The  zone  surrounding  it,  extending  to  the 
periphery,  is  somewhat  cloudy,  and  is  simply  liquefied 
gelatin.  There  is  a  marked  tendency  for  the  liquefac- 
tion to  spread  laterally  and  for  the  colonies  to  run 
together,  so  that,  even  on  plates  containing  few  colonies, 
in  sixty  to  seventy-two  hours  at  from  20°  to  22°  C.,  the 
entire  gelatin  is  usually  converted  into  a  yellowish- 
white  fluid.  Under  these  conditions  growth  is  ac- 
companied by  a  marked  aromatic  odor,  impossible  to 


432 


BACTERIOLOGY. 


describe;  this  is  especially  the  case  when  liquefaction 
is  far  advanced. 


FIG.  79. 


a  b  c  d 

Stab-culture  of  the  Finkler-Prior  bacillus  in  gelatin,  at  18°  to  20°  C. 
a.  After  twenty-four  hours.    6.  After  forty-eight  hours,    c.  After  seventy- 
two  hours,    d.  After  ninety-six  hours. 

In  stab-cultures  in  gelatin  at  the  room-temperature, 
liquefaction  is  noticed  about  the  upper  part  of  the 
needle-track  in  twenty-four  hours ;  this  gradually  in- 
creases, until  at  the  expiration  of  two  or  three  days 
the  entire  upper  portion  of  the  gelatin  has  become 
converted  into  a  cloudy  fluid,  whereas  at  the  lower 
part  of  the  canal  the  liquefaction  progresses  less  rap- 
idly, but  is  still  much  more  marked  than  that  seen  as 
a  result  of  the  growth  of  Koch's  spirillum.  Indeed, 


VIBRIO  PROTEUS.  433 

under  these  circumstances  there  is  no  similarity  what- 
ever between  the  growth  of  the  two  organisms.  (See 
a,  6,  c,  d,  Fig.  79,  and  compare  them  with  correspond- 
ing cuts  in  Fig.  76.) 

Ragged,  more  or  less  dense  masses,  fragments  of  the 
colony  proper,  are  usually  seen  scattered  through  the 
cloudy  liquefied  gelatin. 

On  nutrient  agar-agar  there  is  nothing  particularly 
characteristic  about  its  growth,  appearing  only  as  a 
moist,  grayish  or  yellowish-gray  deposit. 

On  potato  a  pale,  yellowish-gray  deposit  appears ; 
after  forty-eight  to  seventy-two  hours  this  becomes 
moist,  glazed,  and  marked  by  lobulations,  and  sur- 
rounded by  an  irregular,  colorless  zone  of  growth 
that  is  much  less  moist  than  that  forming  the  central 
area.  It  grows  well  on  potato  at  ordinary  room-tem- 
perature. 

It  causes  liquefaction  of  solidified  blood-serum  and 
of  coagulated  egg-albumin. 

In  milk  to  which  neutral  litmus  tincture  has  been 
added  the  blue  color  assumes  a  pink  tinge  in  from  two 
to  three  days  at  37°  to  38°  C. 

It  does  not  form  indol. 

It  does  not  cause  fermentation  of  glucose. 

In  peptone  solution  containing  rosolic  acid  the  color 
is  somewhat  deepened  after  four  or  five  days  at  37°  C. 

EXPERIMENTS  UPON  ANIMALS. — The  ordinary  meth- 
ods of  inoculation  show  this  organism  to  be  without 
pathogenic  properties.  Injections,  subcutaneous  and 
intravascular  and  directly  into  the  stomach,  give  nega- 
tive results.  When  introduced  into  the  stomach  of 
guinea-pigs  by  the  method  employed  by  Koch  in  his 
cholera  experiments,  Finkler  and  Prior  had  3  out  of 

28 


434  BACTERIOLOGY. 

10  animals,  and  Koch  5  out  of  15  animals  so  treated 
to  die. 

In  1885,  1886,  and  1887  Franck1  examined  seven 
cases  that  clinically  presented  the  condition  of  cholera 
nostras;  in  none  of  these  seven  cases  was  the  organism 
of  Finkler  and  Prior,  which  they  claimed  to  be  the 
cause  of  the  disease,  found.  In  all  cases  the  results  of 
bacteriological  examination,  in  so  far  as  the  constant 
presence  of  an  organism  that  might  stand  in  causal 
relation  to  the  disease  was  concerned,  Avere  negative. 
Only  the  ordinary  intestinal  bacteria  were  detected. 

SPIRILLUM  TYROGENUM  (CHEESE  SPIRILLUM  OF 
DENEKE). 

Another  spiral  form,  likewise  forming  short,  comma- 
shaped  segments  in  the  course  of  its  growth  (Fig.  80), 
is  that  found  by  Deneke  in  old  cheese.  It  is  a  little 
smaller  than  Koch's  spirillum.  It  is  motile  and  has 
but  a  single  flagellum,  attached  to  one  of  its  ends.  It 
liquefies  gelatin  more  rapidly  than  does  Koch's  organ- 
ism. It  possesses  no  characteristic  grouping,  as  can  be 
seen  in  impression  cover-slips  of  its  colonies.  It  does 
not  form  spores.  On  gelatin  plates  its  colonies  develop 
very  rapidly  as  saucer-shaped  depressions  ;  after  twenty- 
four  hours  they  vary  from  1  to  4  mm.  in  transverse 
diameter.  To  the  naked  eye  they  are  almost  trans- 
parent, and  are  usually  marked  by  a  denser  centre  and 
peripheral  zone,  the  space  between  being  quite  clear. 
They  are  not  regularly  round  in  all  cases.  A  peculiar 
aromatic  odor  accompanies  their  growth  on  gelatin. 
Under  low  magnifying  power  the  smallest  colonies  are 
irregularly  round  in  outline,  their  borders  being  often 

1  Zeitschrift  fur  Hygiene,  1888,  Band  iv.  S.  207. 


SPIRILLUM  TYROGENUM.  435 

rough  and  broken,  and  the  body  of  the  colony  is  fre- 
quently marked  by  creases  or  ridges  that  give  it  a 
lobulated  appearance.  The  larger  colonies  under  the 


Deneke's  cheese  spirillum,  spirillum  tyrogenum.    From  agar-agar  culture 
twenty-four  hours  old. 

same  lens  appear  as  granular  patches,  a  little  denser  at 
the  periphery  and  centre  than  at  the  intermediate  por- 
tions. The  periphery  gradually  fades  away  and  no 

FIG.  81. 


Colony  of  spirillum  tyrogenum  on  gelatin,  twenty-four  hours  old. 

distinct  circumference  can  be  made  out.  (See  Fig.  81.) 
The  colonies  of  an  intermediate  size,  about  which  lique- 
faction is  just  beginning  to  be  apparent,  show  a  dense 
granular  centre,  the  colony  itself,  and  round  about  it 
a  delicate,  granular  developmental  zone. 

In  stab-cultures  in  gelatin  liquefaction  is  rapid,  caus- 
ing at  the  end  of  twenty-four  hours  a  cup-shaped  depres- 
sion at  the  top  of  the  needle-track,  the  superficial  area 


436 


BACTERIOLOGY. 


of  which  is  about  half  that  of  the  gelatin  in  the  tube. 
(Fig.  82,  a.)  The  liquefying  process  spreads  laterally, 
and  at  the  end  of  forty-eight  hours  the  whole  upper 
portion  of  the  gelatin  may  have  become  liquid.  (Fig. 
82,  b.)  This  process  continues  along  the  track  of  the 


FIG.  82. 


a  bed 

Stab-culture  of  Deneke's  cheese  spirillum  in  gelatin,  at  18°  to  20°  C. 

a.  After  twenty- four  hours.    6.  After  forty-eight  hours,    c.  After  seventy-two 

hours,    d.  After  ninety-six  hours. 

needle,  and  after  seventy-two  and  ninety-six  hours  the 
appearances  shown  in  Fig.  82,  c  and  c7,  will  be  produced. 

There  is  nothing  particularly  characteristic  about  its 
growth  upon  agar-agar. 

On  potato  a  moist,  glazed,  yellowish,  and,  at  points, 


MILLER'S  SPIRILLUM.  437 

brownish-yellow  growth  appears  that  is  surrounded  by  a 
drier,  colorless  zone.     It  is  not  lobulated. 

In  milk  containing  neutral  litmus  tincture  a  pink  color 
appears  after  two  to  three  days  at  37°  C. ;  after  four 
days  the  milk  is  almost  decolorized  and  coagulation  of 
the  casein  is  beginning,  with  a  layer  of  clear  whey  above 
it.  During  the  subsequent  twenty-four  hours  complete 
separation  of  the  contents  of  the  tube  into  clot  and  whey 
takes  place. 

In  Dunham's  peptone  solution  it  does  not  form  indol, 
and  the  reaction  for  this  body  does  not  appear  with 
either  sulphuric  acid  alone  or  plus  sodium  nitrite. 

It  causes  liquefaction  of  both  coagulated  blood-serum 
and  egg-albumin. 

There  is  no  pellicle  formed  as  a  result  of  its  growth 
in  bouillon. 

It  does  not  produce  fermentation  of  glucose. 

In  rosolic-acid-peptone  solution  its  growth  causes  in- 
tensification of  the  red  color  in  four  or  five  days  at 
37°  C. 

By  Koch's  method  of  introducing  cultures  into  the 
stomach  of  a  guinea-pig  this  organism  produced  the 
death  of  three  out  of  fifteen  animals  experimented  upon 
— the  deaths  being  due  most  probably  to  the  toxic  action 
of  the  products  of  growth  that  were  introduced  with  the 
organisms  rather  than  to  any  pathogenic  powers  possessed 
by  the  organism  itself. 


Another  spirillum  that  has  been  likened  to  that  of 
Koch  is  the  one  obtained  by  Miller  from  a  carious  tooth. 
It  has  so  many  characteristics  in  common  with  the  or- 
ganism of  Fiukler  and  Prior  that  Miller  was  inclined 


438  BACTERIOLOGY. 

to  consider  them  identical.      In  morphology  they  are 
indistinguishable.     (See  Fig.  83.)      It  grows  rapidly, 


FIG.  83. 


Spirillum  of  Miller.    From  agar-agar  culture  twenty-four  hours  old. 

.and,  like  the  spirillum  of  Finkler  and  Prior,  causes 
rapid  liquefaction  of  gelatin,  with  the  coincident  pro- 
duction of  a  peculiar  aromatic  odor. 

The  colonies  on  gelatin  plates  appear  after  twenty- 
four  hours  as  small,  transparent  pits  of  liquefaction,  in 
the  centre  of  which  can  be  seen  a  minute  white  point, 
the  colony  itself.  Under  a  low  lens  the  largest  of  these 
points  are  uniformly  granular  and  regularly  round, 
and,  as  a  rule,  are  surrounded  by  a  peripheral  zone  a 
little  darker  than  the  central  portion  of  the  colony. 
The  circumference  is  delicately  fringed  with  short,  cilia- 
like  prolongations  of  growth  which  are  not,  as  a  rule, 
straight,  but  are  twisted  in  all  directions  and  can  only 
be  detected  upon  very  careful  examination.  (See  a, 
Fig.  84.)  When  located  deep  in  the  gelatin  the  colonies 
are  round,  sharply  circumscribed,  of  a  pale  yellowish  or 
greenish-yellow  color,  and  marked  by  very  delicate  irreg- 
ular lines  or  ridges.  After  forty-eight  hours  the  plate 
containing  many  colonies  is  entirely  liquefied,  while  that 
containing  only  a  few  shows  the  presence  of  round, 
sharply-cut,  shallow  pits  of  liquefaction  that  measure 
from  2  to  10  mm.  in  diameter.  They  are  a  little  denser 


MILLER1  S  SPIRILLUM.  439 

at  the  centre  than  at  the  periphery,  and  the  dense  centre 
is  not  sharply  circumscribed,  but  fades  off  into  what  has 
the  appearance  of  a  delicate  film.  (See  6,  Fig.  84.)  As 

FIG.  84. 

..•£  v\w  :£••;:•* . 

i 


6 

Colonies  of  Miller's  spirillum  on  gelatin,  at  20°  to  22°  C.    x  about  57 
diameters. 

a.  Colony  just  beneath  the  surface  of  the  gelatin,    b.  Colony  on  the  surface 
of  the  gelatin. 

the  colonies  become  older  they  are  sometimes  marked 
by  irregular  radii  extending  from  periphery  to  centre, 
like  the  spokes  of  a  wheel. 

In  stab-cultures  in  gelatin  it  rapidly  produces  lique- 
faction, both  at  the  surface  and  along  the  needle-track, 
and  in  most  respects  gives  rise  to  a  condition  very  like 
that  resulting  from  the  growth  of  Finkler  and  Prior's 
spirillum,  though  differing  from  it  in  certain  details. 
(See  a,  6,  c,  d,  Fig.  85.) 

On  agar-agar  nothing  of  special  interest  appears  as  a 
result  of  its  development. 

On  potato  its  growth  is  very  like  that  of  the  cholera 
spirillum,  viz.,  it  appears  at  37°  C.  as  a  dry,  white  patch 
that  lies  quite  flat  upon  the  surface  and  can  often  only 
be  seen  when  the  tube  is  held  to  the  light  at  a  particu- 
lar angle. 

Its  growth  in  bouillon  is  not  characteristic.  It  does 
not  form  a  pellicle. 

It  causes  liquefaction  of  both  coagulated  blood-serum 
and  egg-albumin. 


440 


BACTERIOLOGY. 


It  does  not  produce  indol. 

It  does  not  cause  fermentation  of  glucose. 

It  is  non-motile. 

In  milk  containing  blue  litmus  tincture  it  causes 
almost  complete  decolorization  in  from  three  to  four 
days  at  37°  C.,  with  coincident  coagulation  of  the 


FIG.  85. 


:_ 

«  1>  c  d 

Stab-culture  of  Miller's  spirillum  in  gelatin,  at  18°  to  20°  C. 

a.  After  twenty-four  hours.    6.  After  forty-eight  hours,    c.  After  seventy-two 

hours,    d.  After  ninety-six  hours. 

casein   and   the   formation   of   a  layer  of  clear  whey 
about  it. 

It  causes  the  red  color  of  rosolic-acid-peptone  solu- 
tion to  become  somewhat  intensified  after  four  or  five 
hours  at  37°  C, 


VIBRIO  METCHNIKOVI.  441 

Of  twenty-one  animals  treated  with  this  organism  by 
Koch's  method  of  inoculation  only  four  died. 


VIBRIO   METCHNIKOVI. 


The  spirillum  that  simulates  very  closely  the  comma 
bacillus  of  cholera  in  its  morphological  and  cultural 
peculiarities,  but  which  is  still  easily  distinguished  from 
it,  is  that  described  by  Gamaleia l  under  the  name  of 


FIG.  86. 


Vibrio  Metchnikovi  from  agar-agar  culture,  twenty-four  hours  old. 

vibrio  Metchnikovi.  It  was  found  post  mortem  in  a  num- 
ber of  fowls  that  had  died  in  the  poultry-market  of 
Odessa,  and  the  experiments  of  the  discoverer  led  him 
to  believe  that  it  was  related  etiologically  to  the  gastro- 
enteritis from  which  the  chickens  had  been  suffering. 

Morphologically  it  appears  as  short,  curved  rods  and  as 
longer,  spiral-like  filaments.  It  is  usually  thicker  than 
Koch's  spirillum  and  is  at  times  much  longer,  while 
again  it  is  seen  to  be  shorter.  It  is  usually  more  dis- 
tinctly curved  than  the  "  comma  bacillus/'  (Fig.  86.) 

It  is  supplied  with  a  single  flagellum  at  one  of  its 
extremities,  and  is  therefore  motile. 

It  does  not  form  spores. 

It  is  aerobic. 

Its  growth  upon  gelatin  plates  is  usually  character- 

1  Annales  de  1'Institut  Pasteur,  1888,  tome  ii.,  pp.  482,  552. 


442  BACTERIOLOGY. 

ized,  according  to  PfeifFer,  by  the  appearance  of  two 
kinds  of  liquefying  colonies,  one  strikingly  like  those 
of  the  Finkler-Prior  organism,  the  other  very  similar 
to  those  produced  by  Koch's  comma  bacillus,  though  in 
both  cases  the  liquefaction  resulting  from  the  growth  of 
this  organism  is  more  energetic  than  that  common  to 
the  spirillum  of  Asiatic  cholera.  After  from  twenty- 
four  to  thirty  hours  the  medium-size  colonies,  when 
examined  under  a  low  power  of  the  microscope,  show  a 
yellowish-brown,  ragged  central  mass  surrounded  by  a 
zone  of  liquefaction  that  is  marked  by  a  border  of  deli- 
cate radii.  (Fig.  87.) 

FIG.  87. 


Colony  of  vibrio  Metchnikovi  in  gelatin,  after  thirty  hours  at  20°  to  22°  C. 
X  about  75  diameters. 

In  gelatin  stab-cultures  the  growth  has  much  the 
same  general  appearance  as  that  of  the  cholera  spiril- 
lum, but  is  exaggerated  in  degree.  The  liquefaction 
is  far  more  rapid,  and  the  characteristic  appearance 
of  the  growth  is  lost  in  from  three  to  four  days. 
(See  a,  6,  c,  d,  Fig.  88.)  Development  and  liquefaction 
along  the  deeper  parts  of  the  needle-track  are  much 
more  pronounced  than  is  the  case  with  the  "  comma 
bacillus." 

Its  growth  on  agar-agar  is  rapid ;  after  twenty-four 
to  forty-eight  hours  a  grayish  deposit  appears,  which  has 
a  tendency  to  become  yellowish  with  age. 

On  potato  at  37°  C.  its  growth  is  seen  as  a  moist, 
coffee-colored  patch,  surrounded  by  a  much  paler  zone. 


VIBRIO  METCHNIKOVI. 


443 


The  whole  growth   is  so  smooth  and  glistening  that  it 
has  somewhat  the  appearance  of  being  varnished. 

In  bouillon  it  quickly  causes  opacity,  with  the  ulti- 
mate production  of  a  delicate  pellicle  upon  the  surface. 


FIG.  88. 


a  b  c  d 

Stab-culture  of  vibrio  Metchnikovi  in  gelatin,  at  18°  to  20°  C. 

a.  After  twenty-four  hours,    b.  After  fortj'-eight  hours,  c.  After  seventy-two 
hours,    d.  After  ninety-six  hours. 

It  causes  liquefaction  of  blood-serum,  the  liquefied 
area  being  covered  by  a  dense,  wrinkled  pellicle. 

When  grown  in  peptone  solution  it  produces  indol 
and  coincident!  v  nitrites,  so  that  the  rose-colored  reac- 
tion characteristic  of  indol  is  obtained  by  the  addi- 


444  BACTERIOLOGY. 

tion  of  sulphuric  acid  alone.  The  production  of  indol 
by  this  organism  is  usually  greater  than  that  com- 
mon to  the  comma  bacillus  under  the  same  circum- 
stances. 

In  milk  it  causes  an  acid  reaction  with  coagulation  of 
the  casein.  The  coagulated  casein  collects  at  the  bot- 
tom of  the  tube  in  irregular  masses,  above  which  is  a 
layer  of  clear  whey.  If  blue  litmus  has  been  added 
to  the  milk,  the  color  is  changed  to  pink  in  from 
twenty-four  to  thirty  hours,  and  after  forty-eight  hours 
decolorization  and  coagulation  occur.  The  clots  of 
casein  are  not  re-dissolved.  After  about  a  week  the 
acidity  of  the  milk  is  at  its  maximum,  and  the  organ- 
isms quickly  die. 

It  causes  the  red  color  of  the  rosolic-acid-peptone 
solution  to  become  very  much  deeper  after  four  or  five 
days  at  37°  C. 

It  does  not  cause  fermentation  of  glucose  with  pro- 
duction of  gas. 

It  is  killed  in  five  minutes  by  a  temperature  of  50°  C. 
(Sternberg.) 

It  is  pathogenic  for  chickens,  pigeons,  and  guinea- 
pigs.  Rabbits  and  mice  are  affected  only  by  very  large 
doses. 

Gamaleia  states  that  chickens  affected  with  the  chol- 
eraic gastro-enteritis  of  which  this  organism  is  the  cause, 
are  usually  seen  sitting  quietly  with  ruffled  feathers. 
They  suffer  from  diarrhoea,  but  there  is  no  elevation 
of  temperature.  Hypersemia  of  the  entire  gastro-intes- 
tinal  tract  is  seen  at  autopsy.  The  other  internal  organs 
do  not,  as  a  rule,  present  anything  abnormal  to  the 
naked  eye.  The  intestinal  canal  contains  yellowish 
fluid  with  which  blood  may  be  mixed.  In  adult  chickens 


VIBRIO  METCHNIKOVL  445 

the  spirilla  are  not  found  in  the  blood,  but  in  young 
ones  they  are  usually  present  in  small  numbers. 

After  the  introduction  into  the  pectoral  muscle  of  a 
very  small  quantity  of  a  culture  of  this  organism 
pigeons  succumb  in  from  eight  to  twenty  hours.  The 
most  conspicuous  post-mortem  lesion  is  found  at  the  site 
of  inoculation.  The  muscle  is  marked  by  yellow, 
necrotic  stripes ;  is  more  or  less  cedematous ;  is  swollen, 
and  contains  the  vibrios  in  enormous  numbers.  The 
intestines  are  usually  filled  with  fluid  contents,  which 
may  or  may  not  be  blood-stained ;  the  walls  of  the  in- 
testines are  often  injected  with  blood,  and  occasionally 
markedly  so.  The  conditions  of  the  other  internal 
viscera  are  inconstant.  In .  fatal  cases  the  vibrios  are 
present  in  large  numbers  in  the  blood  and  internal 
organs.  In  pigeons  that  survive  inoculation  the  organ- 
isms may  be  found  only  at  the  site  of  inoculation,  or 
very  sparingly  in  the  blood  also.  These  animals  usually 
exhibit  immunity  from  subsequent  inoculations.  In 
certain  instances  the  results  of  infection  are  chronic; 
the  inoculated  pectoral  muscle  atrophies,  the  pigeon  loses 
in  weight  and  finally  dies  after  one  or  two  weeks.  In 
these  cases  the  organisms  are  usually  absent  from  the 
blood  and  internal  organs,  and  may  even  be  absent  from 
the  site  of  inoculation,  or,  if  present,  in  only  very  small 
number. 

Guinea-pigs  usually  die  in  from  twenty  to  twenty- 
four  hours  after  subcutaneous  inoculation.  At  autopsy 
an  extensive  oadema  of  the  subcutaneous  tissues  about 
the  seat  of  inoculation  is  seen,  and  there  is  usually 
a  necrotic  condition  of  the  tissues  in  the  vicinity  of  the 
point  of  puncture.  As  the  blood  and  internal  organs 
of  both  pigeons  and  guinea-pigs  contain  the  vibrios  in 


446  BACTERIOLOGY. 

large  numbers,  the  infection  in  these  animals  takes, 
therefore,  the  form  of  acute,  general  septicaemia. 

The  blood-serum  of  both  pigeons  and  guinea-pigs 
that  have  survived  inoculation  with  this  organism — i.  e., 
that  have  acquired  immunity  from  it — are  bactericidal 
in  vitrio  for  this  organism.  It  also  possesses  a  certain 
degree  of  immunity-conferring  property,  as  may  be 
demonstrated  by  injecting  it  into  normal  pigeons  and 
guinea-pigs  that  are  subsequently  to  be  inoculated  with 
virulent  cultures. 

Very  old  cultures  of  this  organism  in  bouillon  be- 
come distinctly  alkaline  in  reaction.  At  this  stage  they 
contain  a  toxin  that  is  markedly  active  for  susceptible 
animals.  This  toxin  is  not  dissolved  in  the  fluid  to  any 
extent,  but  is  apparently  in  intimate  association  with  the 
proteid  matters  composing  the  bacteria. 

Gastro-enteritis  may  be  produced  in  both  chickens 
and  guinea-pigs  by  feeding  them  with  food  with  which 
cultures  of  this  organism  have  been  mixed.  (Gamaleia.) 

In  the  autumn  of  1896  the  author  isolated  from  the 
water  of  the  Schuylkill  River  at  Philadelphia  a  spirillum 
that  is  pathogenic  for  pigeons  and  guinea-pigs,  and 
that  possesses  so  many  of  the  other  characteristics 
common  to  the  group  of  spirilla  of  which  the  chol- 
era spirillum  is  the  most  important  member,  as  to  justify 
the  opinion  that  they  are  of  the  same  family.  That  it 
is  not  identical  with  the  cholera  spirillum  is  evident, 
for  the  reason  that  the  latter  produces  cholera,  while 
the  vibrio  Sehuylkilliensis  manifestly  does  not.1 

1  For  the  detailed  description  of  this  organism,  see  Journal  of  Ex- 
perimental Medicine,  vol.  i.  p.  419 ;  also  Transactions  of  the  Association 
of  American  Physicians,  1896,  vol.  xi.  p.  394. 


VIBRIO  SCHUYLKILLIENSIS.  447 

NOTE. — Since  the  epidemic  of  cholera  in  Hamburg 
quite  a  number  of  curved  or  spiral  organisms,  some- 
what like  the  cholera  spirillum,  have  been  discovered. 
For  the  descriptions  of  these  the  reader  is  referred  to 
current  bacteriological  literature. 


CHAPTER  XXIV. 

Study  of  bacillus  anthracis,  and  of  the  effects  produced  by  its  inoculation 
into  animals — Peculiarities  of  the  organism  under  varying  con- 
ditions of  surroundings. 

THE  discovery  that  the  blood  of  animals  suffering 
from  splenic  fever,  or  anthrax,  always  contained  minute 
rod-shaped  bodies  (Pollender,  1855 ;  Davaine,  1863), 
led  to  a  closer  study  of  this  disease,  and  has  resulted 
probably  in  contributing  more  to  our  knowledge  of 
bacteriology  in  general  than  work  upon  any  of  the 
other  infectious  maladies. 

The  outcome  of  these  investigations  is  that  a  rod- 
shaped  micro-organism,  now  known  as  bacillus  an- 
thracis, is  always  present  in  the  blood  of  animals  suffer- 
ing from  this  disease  ;  that  this  organism  can  be  obtained 
from  the  tissues  of  these  animals  in  pure  cultures ;  and 
that  these  artificial  cultures  of  bacillus  anthracis  when 
introduced  into  the  bodies  of  susceptible  animals  can 
again  produce  a  condition  identical  with  that  found  in 
the  animal  from  which  they  were  obtained.  The  dis- 
ease is  a  true  septicaemia,  and  after  death  the  capil- 
laries throughout  the  body  are  always  found  to  contain 
the  typical  rod-shaped  organism  in  larger  or  smaller 
numbers. 

This  organism,  when  isolated  in  pure  culture,  is  a 
bacillus  which  varies  considerably  in  length,  ranging 
from  short  rods  2  to  3  //  in  length  to  longer  threads 
20  to  25  ft  in  length.  In  breadth  it  is  from  1  to 

448 


BACILLUS  ANTHRACIS.  449 

1.25  fJL.     Frequently  very  long  threads,  made   up   of 
several  rods  joined  end  to  end,  are  seen. 

When  obtained  directly  from  the  body  of  an  animal 
it  is  usually  in  the  form  of  short  rods  square  at  the  ends. 
If  highly  magnified,  the  ends  are  seen  to  be  a  trifle 
thicker  than  the  body  of  the  cell  and  somewhat  indented 
or  concave,  peculiarities  that  help  to  distinguish  it  from 
certain  other  organisms  that  are  somewhat  like  it  mor- 
phologically. (See  Fig.  89.) 


FIG.  89. 


s 

Bacillus  anthracis,  highly  magnified  to  show  swellings  and  concavities  at 
extremities  of  the  single  cells. 

When  cultivated  artificially  at  the  temperature  of  the 
body  the  bacillus  of  anthrax  presents  a  series  of  very 
interesting  stages. 

The  short  rods  develop  into  long  threads,  which 
may  be  seen  twisted  or  plaited  together  like  ropes, 
each  thread  being  marked  by  the  points  of  juncture 
of  the  short  rods  composing  it.  (Fig.  90,  a  and  6.) 
In  this  condition  it  remains  until  alterations  in  its  sur- 
roundings, the  most  conspicuous  being  diminution  of  its 
nutritive  supply,  favor  the  production  of  spores.  When 
this  stage  begins  changes  in  the  protoplasm  of  the 
bacilli  may  be  noticed ;  they  become  marked  by  irregu- 
lar, granular  bodies,  which  eventually  coalesce  into 
glistening,  oval  spores,  one  of  which  lies  in  nearly  every 
segment  of  the  long  thread,  and  gives  to  the  thread  the 

29 


450  BACTERIOLOGY. 

appearance  of  a  string  of  glistening  beads.  (Fig.  91.) 
In  this  stage  they  remain  but  a  short  time.  The  chains 
of  spores,  which  are  held  together  by  the  remains  of 


FIG.  90. 


Bacillus  anthracis.    'Plaited  and  twisted  threads   seen  in  fresh  growing 
cultures.    X  about  400  diameters. 

the  cells  in  which  they  formed,  become  broken  up,  and 
eventually  nothing  but  free  oval  spores,  and  here  and 
there  the  remains  of  mature  bacilli  which  have  under- 
gone degenerative  changes,  can  be  found.  In  this  con- 
dition the  spores,  capable  of  resisting  deleterious  influ- 

FlG.  91. 


Threads  of  bacillus  anthracis  containing  spores.    X  about  1200  diameters. 

ences,  remain  and,  unless  their  surroundings  are  altered, 
continue  in  this  living,  though  inactive,  condition  for 
a  very  long  time.  If  again  placed  under  favorable 
conditions,  each  spore  will  germinate  into  a  mature  cell, 


BACILLUS  ANTHRACIS.  451 

and  the  same  series  of  changes  will  be  repeated  until 
the  surroundings  become  again  gradually  unfavorable 
to  development,  when  spore-formation  again  takes  place. 
Spore-formation  occurs  only  at  temperatures  ranging 
from  18°  to  43°  C.,  37.5°  C.  being  the  optimum. 
Under  12°  C.  they  are  not  formed.  This  organism 
does  not  form  spores  in  the  tissues  of  the  living 
animal,  its  usual  condition  at  this  time  being  that  of 
short  rods;  occasionally,  however,  somewhat  longer 
forms  may  be  seen. 

The  bacillus  of  anthrax  is  not  motile. 

GROWTH  ON  AGAR-AGAR. — Colonies  of  this  organ- 
ism, as  seen  upon  agar-agar,  present  a  very  typical 
appearance,  from  which  they  have  been  likened  unto 
the  head  of  Medusa.  From  a  central  point  which  is 

FIG.  92. 


Colony  of  bacillus  anthracis  on  agar-agar. 

more  or  less  dense,  consisting  of  a  felt-like  mass  of 
long  threads  irregularly  matted  together,  the  growth 
continues  outward  upon  the  surface  of  the  agar-agar. 
(Fig.  92.)  It  is  made  up  of  wavy  bundles  in  which 
the  threads  are  seen  to  lie  parallel  or  are  twisted  in 
strands  like  those  of  a  rope ;  sometimes  they  have  a 
plaited  arrangement.  (See  Fig.  90.)  These  bundles 
twist  and  cross  in  all  directions,  and  eventually  dis- 


452  BACTERIOLOGY. 

appear  at  the  periphery  of  the  colony.  At  the  ex- 
treme periphery  of  the  colonies  it  is  sometimes  possible 
to  trace  single  bundles  of  these  threads  for  long  dis- 
tances across  the  surface  of  the  agar-agar.  The  colony 
itself  is  not  circumscribed  in  appearance,  but  is  more 
or  less  irregularly  fringed  or  ragged,  or  scalloped.  To 
the  naked  eye  they  look  very  much  like  minute  pellicles 
of  raw  cotton  that  have  been  pressed  into  the  surface 
of  the  agar-agar. 

As  the  colonies  continue  to  grow  they  become  more 
and  more  dense  and  opaque,  and  granular  and  rough  on 
the  surface.  When  touched  with  a  sterilized  needle 
one  experiences  a  sensation  that  suggests  somewhat 
their  matted  structure.  They  are  never  moist  or 
creamy.  The  bit  that  is  taken  up  with  the  needle  is 
always  more  or  less  ragged. 

GELATIN. — The  colonies  on  gelatin  at  the  earliest 
stages  also  present  the  same  wavy  appearance  ;  but  this 
characteristic  soon  becomes  in  part  destroyed  by  the 
liquefaction  of  the  gelatin  which  is  produced  by  the 
growing  organisms.  This  allows  them  to  sink  to  the 
bottom  of  the  fluid,  where  they  lie  as  an  irregular  mass. 
Through  the  fluid  portion  of  the  gelatin  may  be  seen 
small  clumps  of  growing  bacilli,  which  look  very  much 
like  bits  of  cotton-wool. 

BOUILLON. — In  bouillon  the  growth  is  characterized 
by  the  formation  of  flaky  masses,  which  also  have  very 
much  the  appearance  of  bits  of  raw  cotton.  Micro- 
scopic examination  of  one  of  these  flakes  reveals  the 
twisted  and  plaited  arrangement  of  the  long  threads. 

POTATO. — On  this  medium  it  develops  rapidly  as  a 
dull,  dry,  granular,  whitish  mass,  which  is  more  or  less 
limited  to  the  point  of  inoculation.  On  potato,  at  the 


BACILLUS  ANTHRACIS.  453 

temperature  of  the  incubator,  spore-formation  may  be" 
easily  observed. 

STAB-  AND  SLANT-CULTURES. — Stab-  and  slant-cult- 
ures on  agar-agar  present  in  general  the  appearances 
given  for  the  colonies,  except  that  the  growth  is  much 
more  extensive.  The  growth  is  always  more  pro- 
nounced on  the  surface  than  down  the  track  of  the 
needle. 

On  gelatin  it  causes  liquefaction,  which  begins  on  the 
surface  at  the  point  inoculated  and  spreads  outward  and 
downward. 

It  grows  best  with  access  to  oxygen,  and  very  poorly 
when  the  supply  of  that  gas  is  interfered  with. 

Under  favorable  conditions  of  aeration,  nutrition,  and 
temperature  its  growth  is  rapid. 

Under  12°  C.  and  above  45°  C.  no  growth  occurs. 
Its  optimum  temperature  is  that  of  the  human  body, 
viz.,  37°-38°  C. 

The  spores  of  the  anthrax  bacillus  are  very  resistant  to 
heat,  though  the  degree  of  resistance  varies  with  spores 
of  different  origin.  Von  Esmarch  found  that  anthrax 
spores  from  some  sources  were  readily  killed  by  an  ex- 
posure of  one  minute  to  the  temperature  of  steam, 
whereas  spores  from  other  sources  resisted  this  temper- 
ature longer,  in  some  cases  as  long  as  twelve  minutes. 

STAINING. — Anthrax  bacilli  stain  readily  with  the 
ordinary  aniline  dyes.  In  tissues  their  presence  may 
also  be  demonstrated  by  the  ordinary  aniline  stain- 
ing-fluids  or  by  Gram's  method.  They  may  also  be 
stained  in  tissues  with  a  strong  watery  solution  of 
dahlia,  after  which  the  sections  are  decolorized  in  2  per 
cent,  sodium  carbonate  solution,  washed  in  water,  dehy- 
drated in  alcohol,  cleared  in  xylol,  and  mounted  in  bal- 


454  BACTERIOLOGY. 

sam.  This  leaves  the  bacilli  stained,  while  the  tissues 
containing  them  are  decolorized ;  or  the  latter  maybe 
stained  a  contrast-color — with  eosin,  for  example — after 
dehydration  in  alcohol  and  before  clearing  in  xylol.  In 
this  case  they  must  be  washed  again  in  alcohol  before 
using  the  xylol.  In  a  preparation  treated  in  this  way 
the  rod-shaped  organisms  are  of  a  purple  color,  and  will 
be  seen  in  the  capillaries  of  the  tissues,  while  the  tissues 
themselves  are  of  a  pale  rose  color. 

INOCULATION  INTO  ANIMALS. — Introduce  into  the 
subcutaneous  tissues  of  the  abdominal  wall  of  a  guinea- 
pig  or  rabbit  a  portion  of  a  pure  culture  of  bacillus 
anthrads.  The  animal  usually  succumbs  in  from  thirty- 
six  to  forty-eight  hours.  Little  or  no  reaction  at  the 
immediate  point  of  inoculation  will  be  noticed ;  but 
beyond  this,  extending  for  a  long  distance  over  the 
abdomen  and  thorax,  the  tissues  will  be  markedly 
O3dematous.  Here  and  there,  scattered  through  this 
oadematous  tissue,  small  ecchymoses  will  be  seen. "  The 
underlying  muscles  are  pale  in  color.  Inspection  of  the 
internal  viscera  reveals  no  very  marked  macroscopic 
changes  except  in  the  spleen.  This  is  enlarged,  dark 
in  color,  and  soft.  The  liver  may  present  the  appear- 
ance of  cloudy  swelling ;  the  lungs  may  be  red  or  pale 
red  in  color ;  the  heart  is  usually  filled  with  blood.  No 
other  changes  can  be  seen  by  the  naked  eye. 

Prepare  cover-slip  preparations  from  the  blood  and 
other  viscera.  They  will  all  be  found  to  contain  short 
rods  in  large  numbers.  Nowhere  can  spore-formation 
be  detected.  Upon  microscopic  examination  of  sec- 
tions of  the  organs  which  have  been  hardened  in 
alcohol  the  capillaries  are  seen  to  be  filled  with  the 
bacilli ;  in  some  places  closely  packed  in  large  num- 


BACILLUS  ANTHRACIS.  455 

bers,  at  other  points  fewer  in  number.  Usually 
they  are  present  in  largest  numbers  in  those  tis- 
sues having  the  greatest  capillary  distribution  and  at 
those  points  at  which  the  circulation  is  slowest.  They 
are  uniformly  distributed  through  the  spleen.  The 
glomeruli  of  the  kidneys  and  the  capillaries  of  the 

FIG.  93. 


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\ 

Anthrax  bacilli  in  liver  of  mouse.    X  about  450  diameters.    Bacilli  stained 
by  Gram's  method ;  tissue  stained  with  Bismarck-brown. 

lungs  are  frequently  packed  with  them.  The  capillaries 
of  the  liver  contain  them  in  large  numbers.  (Fig.  93.) 
Hemorrhages,  probably  due  to  rupture  of  capillaries 
by  the  mechanical  pressure  of  the  bacilli  which  are 
developing  within  them,  not  uncommonly  occur. 
When  these  occur  in  the  mucous  membranes  of  the 
alimentary  tract  the  blood  may  escape  through  the 
mouth  or  anus ;  when  in  the  kidneys,  through  the  uri- 
niferous  tubules. 

Cultures  from  the  different  organs  or  from  the  oedema- 
tous  fluid  about  the  point  of  inoculation  result  in  growth 
of  bacillus  anthracw. 

The  amphibia,  dogs,  and  the  majority  of  birds  are 
not  susceptible  to  this  disease.  Eats  are  difficult  to 


456  BACTERIOLOGY. 

infect.  Rabbits,  guinea-pigs,  white  mice,  gray  house- 
mice,  sheep,  and  cattle  are  susceptible.  Infection  may 
occur  either  through  the  circulation,  through  the  air- 
passages,  through  the  alimentary  tract,  or,  as  we  have 
just  seen,  through  the  subcutaneous  tissues. 

PROTECTIVE   INOCULATION. 

The  most  noteworthy  application  of  artificially  pre- 
pared living  vaccines  to  the  protection  of  animals 
against  infection  is  seen  in  connection  with  anthrax 
in  sheep  and  in  bovines. 

By  a  variety  of  procedures  the  virulent  anthrax 
bacillus  may  be  in  part  or  totally  robbed  of  its  patho- 
genic properties.  It  is  through  the  very  mild  consti- 
tutional disturbance  caused  in  animals  vaccinated  with 
such  weakened  cultures  that  protection  is  often  afforded 
against  the  severer,  frequently  fatal,  form  of  the  infec- 
tion. 

Without  reviewing  the  various  methods  that  have 
been  employed  for  attenuating  the  virulence  of  this 
organism  to  a  degree  suitable  for  protective  vaccina- 
tion, it  will  suffice  to  say  that  the  most  satisfactory 
results  have  been  obtained  by  long-continued  cultiva- 
tion (ten  to  thirty  days)  at  a  temperature  of  from  42° 
to  43°  C.  In  this  procedure  the  spore-free,  virulent 
bacillus  anthracis,  obtained  directly  from  the  blood  of 
a  recently  dead  animal,  is  brought  at  once  into  sterile 
nutrient  bouillon  in  about  twenty  test-tubes,  which  are 
immediately  placed  in  an  incubator  that  is  carefully 
regulated  to  maintain  a  temperature  of  42.5°  C.  There 
should  not  be  a  fluctuation  of  over  0.1°  C. 

After  about  a  week  a  tube  is  removed  from  the  incu- 
bator on  each  successive  day  and  its  virulence  tested  at 


BACILLUS  ANTHRACIS.  457 

once  on  animals.  The  degree  of  attenuation  experi- 
enced by  the  cultures  grown  under  these  circumstances 
is  determined  by  tests  upon  rabbits,  guinea-pigs,  and 
mice.  The  first  culture  removed  may  or  may  not 
kill  rabbits,  the  most  resistant  of  the  three  animals 
used  for  the  test,  while  it  will  certainly  kill  the  guinea- 
pigs  and  mice ;  after  another  two  or  three  days  rabbits 
will  no  longer  succumb  to  inoculation  with  the  culture 
last  removed  from  the  incubator,  while  no  diminution 
will  as  yet  be  noticed  in  its  pathogenesis  for  the  other 
two  species.  After  four  to  seven  days  more  a  culture 
may  be  encountered  that  kills  only  mice,  the  guinea- 
pigs  escaping;  while  ultimately,  if  the  experiment  be 
continued,  a  degree  of  attenuation  may  be  reached  in 
which  the  organism  has  not  even  the  power  of  killing 
a  mouse,  though  it  still  retains  its  vitality.  Investiga- 
tion of  these  attenuations  shows  them  to  possess  all 
the  characteristics  of  enfeebled  anthrax  bacilli ;  they 
grow  slowly  and  less  vigorously  when  transplanted  ; 
they  do  not  form  spores  when  exposed  to  a  high  tem- 
perature ;  and  microscopically  they  present  evidences 
of  degeneration.  When  introduced  beneath  the  skin 
of  animals  they  disseminate  but  slightly  beyond  the 
site  of  inoculation,  and  do  not,  as  a  rule,  cause  the 
general  septicaemia  that  occurs  in  susceptible  animals 
after  inoculation  with  normal  cultures  of  this  organism. 
In  the  practical  employment  of  these  attenuated  cult- 
ures for  protective  purposes  two  vaccines  are  employed. 
These  were  designated  by  Pasteur  as  "first"  and 
"  second  "  vaccines.  The  "  first "  is  the  one  that  killed 
only  the  mice  in  the  preliminary  tests;  while  the  "sec- 
ond "  is  that  which  killed  both  mice  and  guinea-pigs, 
but  failed  to  kill  the  rabbit.  When  larger  animals, 


458  BACTERIOLOGY. 

such  as  sheep  or  cattle,  are  to  be  protected  by  vaccina- 
tion with  these  vaccines,  a  subcutaneous  inoculation  of 
about  0.3  c.c.  of  the  first  vaccine  is  usually  given. 
This  should  be  practically  without  noticeable  effect, 
causing  neither  rise  of  body-temperature  nor  other 
constitutional  or  local  symptoms.  After  a  period  of 
about  two  weeks  the  second  vaccine  is  injected  in  the 
same  way ;  this  may  or  may  not  cause  disturbance. 
In  the  event  of  its  doing  so  the  symptoms  are  rarely 
alarming,  and,  if  the  vaccines  have  been  properly  pre- 
pared and  tested  before  use,  they  disappear  within  a 
short  time  after  the  injection. 

In  the  large  majority  of  cases  sheep,  bovines,  horses, 
and  mules  may  be  safely  protected  against  anthrax  by 
the  careful  practice  of  this  method. 

EXPERIMENTS. 

Prepare  three  cultures  of  anthrax  bacilli — one  upon 
gelatin,  one  upon  agar-agar,  and  one  upon  potato. 
Allow  the  gelatin  culture  to  remain  at  the  ordinary 
temperature  of  the  room,  place  the  agar-agar  culture 
in  the  incubator,  and  the  potato  culture  at  a  temper- 
ature not  above  18°  to  20°  C.  Prepare  cover-slips 
from  each  from  day  to  day.  What  differences  are 
observed  ? 

Prepare  two  potato  cultures  of  the  anthrax  bacillus. 
Place  one  in  the  incubator  and  maintain  the  other  at  a 
temperature  of  from  18°  to  20°  C.  Examine  them 
each  day.  Do  they  develop  in  the  same  way  ? 

From  a  fresh  culture  of  anthrax  bacilli,  in  which 
spore-formation  is  not  yet  begun  (which  is  the  surest 


BACILLUS  ANTHRACIS.  459 

source  from  which  to  obtain  non-spore-bearing  anthrax 
bacilli),  prepare  a  hanging-drop  preparation  ;  also  a 
cover-slip  preparation  in  the  usual  way  and  stain  it 
with  a  strong  gentian-violet  solution ;  and  another 
cover-slip  preparation  which  is  to  be  drawn  through 
a  flame  twelve  to  fifteen  times,  stained  with  aniline 
gentian-violet,  washed  in  iodine  solution  and  then 
in  water.  Examine  these  microscopically.  Do  they 
all  present  the  same  appearance?  To  what  are  the 
differences  due? 

Do  the  anthrax  threads,  as  seen  in  a  fresh,  growing, 
hanging  drop,  present  the  same  morphological  appear- 
ance as  when  dried  and  stained  upon  a  cover-slip? 
How  do  they  differ? 

Liquefy  a  tube  of  agar-agar,  and  when  it  is  at  the 
temperature  of  40°  to  43°  0.  add  a  very  minute  quan- 
tity of  an  anthrax  culture  which  is  far  advanced  in  the 
spore-stage.  Mix  it  thoroughly  with  the  liquid  agar- 
agar  and  from  this  prepare  several  hanging  drops  under 
strict  antiseptic  precautions,  using  the  fluid  agar-agar 
for  the  drops  instead  of  bouillon  or  salt-solution.  Select 
from  among  these  preparations  that  one  in  which  the 
smallest  number  of  spores  are  present.  Under  the 
microscope  observe  the  development  of  a  spore  into  a 
mature  cell.  Describe  carefully  the  developmental 
stages. 

Prepare  a  1  : 1000  solution  of  carbolic  acid  in  bouil- 
lon. Inoculate  this  with  virulent  anthrax  spores.  If 
no  development  occurs  after  two  or  three  days  at  the 
temperature  of  the  thermostat,  prepare  a  solution  of 
1  : 1200,  and  continue  until  the  point  is  reached  at 


460  BACTERIOLOGY. 

which  the  amount  of  carbolic  acid  present  just  permits 
of  the  development  of  the  spores.  When  the  proper 
dilution  is  reached  prepare  a  dozen  of  such  tubes  and 
inoculate  one  of  them  with  virulent  anthrax  spores. 
As  soon  as  development  is  well  advanced  transfer  a 
loopful  from  this  tube  into  a  second  of  the  carbolic  acid 
tubes ;  when  this  has  developed,  then  from  this  into  a 
third,  etc.  After  five  or  six  generations  have  been 
treated  in  this  way  study  the  spore-production  of  the 
organisms  in  that  tube.  If  it  is  normal,  continue  to 
inoculate  from  one  carbolic  acid  tube  to  another,  and 
see  if  it  is  possible  by  this  means  to  influence  in  any 
way  the  production  of  spores  by  the  organism  with 
which  you  are  working.  What  is  the  effect,  if  any? 

Prepare  two  bouillon  cultures,  each  from  one  drop  of 
blood  of  an  animal  dead  of  anthrax.  (Why  from  the 
blood  of  an  animal  and  not  from  a  culture  ?)  Allow  one 
of  them  to  grow  for  from  fourteen  to  eighteen  hours  in 
the  incubator ;  allow  the  other  to  grow  at  the  same  tem- 
perature for  three  or  four  days.  Remove  the  first  tube 
after  the  time  mentioned  and  subject  it  to  a  temperature 
of  80°  C.  for  thirty  minutes.  At  the  end  of  this  time 
prepare  four  plates  from  it.  Make  each  plate  with  one 
drop  from  the  heated  bouillon  culture.  At  the  end  of 
three  or  four  days  treat  the  second  tube  in  identically 
the  same  way.  How  do  the  number  of  colonies  which 
develop  from  the  two  cultures  compare  ?  Was  there 
any  difference  in  the  time  required  for  their  develop- 
ment on  the  plates? 

From  a  potato  culture  of  anthrax  bacilli  which  has 
been  in  the  incubator  for  three  or  four  days  scrape 


BACILLUS  ANTHRACIS.  461 

away  the  growth  and  carefully  break  it  up.  in  10  c.c. 
of  sterilized  physiological  salt-solution.  The  more 
thoroughly  it  is  broken  up  the  more  accurate  will  be  the 
results  of  the  experiment.  Place  this  in  a  bath  of  boil- 
ing water,  and  at  the  end  of  one,  three,  five,  seven,  and 
ten  minutes  make  plates  upon  agar-agar  each  with  one 
loopful  of  the  contents  of  this  tube.  Are  the  results 
on  the  plates  alike  ? 

Determine  the  exact  time  necessary  to  sterilize  ob- 
jects, such  as  silk  or  cotton  threads,  on  which  anthrax 
spores  have  been  dried,  by  the  steam  method  and  by 
the  hot-air  method. 

Prepare  a  bouillon  culture  from  the  blood  of  an  ani- 
mal just  dead  of  anthrax.  After  this  has  been  in  the 
incubator  for  from  three  to  four  hours  subject  it  to  a 
temperature  of  55°  C.  for  ten  minutes.  At  the  end 
of  this  time  make  plates  from  it  and  also  inoculate  a 
rabbit  subcutaneously  with  it.  What  are  the  results? 
Are  the  colonies  on  the  plates  in  every  way  charac- 
teristic ? 

Inoculate  six  Erlenmeyer  flasks  of  sterile  bouillon, 
each  containing  about  35  c.c.  of  the  medium,  from 
either  the  blood  of  an  animal  just  dead  of  anthrax  or 
from  a  fresh  virulent  culture  in  which  no  spores  are 
formed. 

Place  these  flasks  in  the  incubator  at  a  temperature 
of  42.5°  C.  At  the  end  of  five,  ten,  fifteen,  twenty, 
twenty-five,  or  more  days  remove  a  flask.  Label 
each  flask  as  it  is  taken  from  the  incubator  with  the 
exact  number  of  days  that  it  has  been  at  the  tempera- 


462  BACTERIOLOGY. 

ture  of  42.5°  C.  Study  each  flask  carefully,  both  in 
its  culture-peculiarities  and  in  its  pathogenic  properties 
when  employed  on  animals. 

Are  these  cultures  identical  in  all  respects  with  those 
that  have  been  kept  at  37°  C.? 

If  they  differ,  in  what  respect  is  the  difference  most 
conspicuous  ? 

Should  any  of  the  animals  survive  the  inoculations 
made  from  the  different  cultures  in  the  foregoing  ex- 
periment, note  carefully  which  one  it  is,  and  after  ten 
to  twelve  days  repeat  the  inoculation,  using  the  same 
culture;  if  it  again  survives,  inoculate  it  with  the  cult- 
ure preceding  the  one  just  used  in  the  order  of  removal 
from  the  incubator ;  if  it  still  survives,  inoculate  it  with 
virulent  anthrax.  What  is  the  result?  How  is  the 
result  to  be  explained  ?  Do  the  cultures  which  were 
made  from  these  flasks  at  the  time  of  their  removal 
from  the  incubators  act  in  the  same  way  toward  animals 
as  the  organisms  growing  in  the  flasks  ?  Is  the  action 
of  each  of  these  cultures  the  same  for  mice,  guinea-pigs, 
and  rabbits? 

Prepare  a  2  per  cent,  solution  of  sulphuric  acid  in 
distilled  water ;  suspend  in  this  a  number  of  anthrax 
spores;  at  the  end  of  three,  six,  and  nine  days  at  35°  C. 
inoculate  both  a  guinea-pig  and  a  rabbit.  Prepare 
cultures  from  this  suspension  on  the  third,  sixth,  and 
ninth  days ;  when  the  cultures  have  developed  inoculate 
a  rabbit  and  a  guinea-pig  from  the  culture  made  on  the 
ninth  day.  Should  the  animals  survive,  inoculate  them 
again  after  three  or  four  days  with  a  culture  made  on 
the  sixth  day.  Do  the  results  appear  in  any  way 
peculiar  ? 


CHAPTER    XXV. 

The  most  important  of  the  organisms  found  in  the  soil — The  nitrify- 
ing bacteria — The  bacillus  of  tetanus — The  bacillus  of  malignant 
oedema — The  bacillus  of  symptomatic  anthrax. 

THE   NITRIFYING   BACTERIA. 

BY  the  employment  of  bacteriological  methods  in 
the  study  of  the  soil  much  light  has  been  shed  upon 
the  cause  and  nature  of  the  interesting  and  momen- 
tous biological  phenomena  there  constantly  in  prog- 
ress. Of  these,  the  one  that  is  of  the  greatest  im- 
portance comprises  those  changes  that  accompany  the 
widespread  process  of  disintegration  and  decomposi- 
tion, to  which  reference  has  already  been  made.  (See 
Chapter  I.)  This  resolution  of  dead  complex  organic 
compounds  into  simpler  structures  that  are  assimilable 
as  food  by  growing  vegetation  is  dependent  upon 
the  activities  of  bacteria  located  in  the  superficial 
layers  of  the  ground.  It  is  not  a  simple  process, 
brought  about  by  a  single,  specific  species  of  bacteria ; 
but  represents  a  series  of  metabolic  alterations,  each 
definite  step  of  which  is  most  probably  the  result  of 
the  activities  of  different  species  or  groups  of  species, 
acting  singly  or  together  (symbiotically).  Our  knowl- 
edge upon  the  subject  is  not  sufficient  to  permit  us  to 
follow  in  detail  the  manifold  alterations  undergone 
by  dead  organic  material  in  the  process  of  decomposi- 
tion that  results  in  its  conversion  into  inorganic  com- 
pounds, with  the  formation  of  carbonic  acid,  ammonia, 
and  water  as  the  conspicuous  end-products.  It  suffices 

463 


464  BACTERIOLOGY. 

to  say  that  wherever  dead  organic  matters  are  exposed 
to  the  action  of  the  great  group  of  saprophytic  bacteria, 
in  which  are  found  many  different  species,  the  altera- 
tions through  which  they  pass  are  ultimately  character- 
ized by  the  appearance  of  these  three  bodies.  When  the 
process  of  decomposition  occurs  in  the  soil,  however,  it 
does  not  cease  at  this  point,  but  we  find  still  further 
alterations  —  alterations  concerning  more  particularly 
the  ammonia.  This  change  in  ammonia  is  character- 
ized by  the  products  of  its  oxidation,  viz.,  by  the  for- 
mation of  nitrous  and  nitric  acids  and  their  salts ;  it  is 
not  a  result  of  the  direct  action  of  atmospheric  oxygen 
upon  the  ammonia,  but  occurs  through  the  instrumen- 
tality of  a  special  group  of  saprophytes  known  as  the 
nitrifying  organisms.  They  are  found  in  the  most  super- 
ficial layers  of  the  ground,  and  though  more  common 
in  some  places  than  in  others,  they  are,  nevertheless, 
present  over  the  entire  surface  of  the  earth.  The 
most  conspicuous  example  of  the  functional  activity  of 
this  specific  form  of  soil  organism  is  seen  in  the  im- 
mense saltpetre-beds  of  Chili  and  Peru,  where,  by  the 
activities  of  these  microscopic  plants,  nitrates  are  pro- 
duced from  the  ammonia  of  the  faecal  evacuations  of 
sea-fowl  in  such  enormous  quantities  as  to  form  the 
source  of  supply  of  this  article  for  the  commercial 
world.  A  more  familiar  example,  though  hardly  upon 
such  a  great  scale,  is  seen  in  the  decomposition  and 
subsequent  nitrification  of  the  organic  matters  of  sew- 
age and  other  impure  waters  in  the  process  of  puri- 
fication by  filtration  through  the  soil,  a  process  in  which 
it  is  possible  to  follow,  by  chemical  means,  the  organic 
matters  from  their  condition  as  such  through  their  con- 
spicuous modifications  to  their  ultimate  conversion  into 


THE  NITRIFYING  BACTERIA.  465 

ammonia,  nitrous  and  nitric  acids.  In  fact,  the  same 
breaking  down  and  building  up,  resulting  ultimately 
in  nitrification,  occurs  in  all  nitrogenous  matters  that 
are  deposited  upon  the  soil  and  allowed  to  decay.  It  is 
largely  through  this  means  that  growing  vegetation 
obtains  the  nitrogen  necessary  for  the  nutrition  of  its 
tissues,  and  when  viewed  from  this  standpoint  we  ap- 
preciate the  importance  of  this  process  to  all  life,  ani- 
mal as  well  as  vegetable,  upon  the  earth. 

These  very  important  and  interesting  nitrifying 
organisms,  of  which  there  appear  to  be  several,  have 
been  the  subject  of  much  study,  and  are  found  to 
possess  peculiarities  of  sufficient  interest  to  justify  a 
more  or  less  detailed  description.  For  a  long  time  all 
efforts  to  isolate  them  from  the  soils  in  which  they  were 
believed  to  be  present,  and  to  cultivate  them  by  the 
processes  commonly  employed  in  bacteriological  work, 
resulted  in  failure ;  and  it  was  not  until  it  was  found 
that  the  ordinary  methods  of  bacteriological  research 
were  in  no  way  applicable  to  the  study  of  these  bacteria 
that  other,  and  ultimately  successful,  methods  were  de- 
vised. By  these  special  devices  nitrifying  bacteria, 
Capable  of  oxidizing  ammonia  to  nitric  acid,  have  been 
isolated  and  cultivated,  and  the  more  important  of  their 
biological  peculiarities  recorded  by  Winogradsky  in 
Switzerland,  by  G.  C.  and  P.  F.  Frankland  in  Eng- 
land, and  by  Jordan  and  Richards  in  this  country. 
From  the  similarity  of  the  properties,  given  by  these 
several  observers,  of  the  nitrifying  organisms  isolated 
by  them,  it  seems  likely  that  they  have  all  been  work- 
ing with  either  the  same  organism  or  very  closely  allied 
species. 

The  organism  generally  known  as  the  nitro-monas  of 

30 


466  BACTERIOLOGY. 

Winogradsky  is  a  short,  oval,  and  frequently  almost 
spherical  cell.  It  divides  as  usual  for  bacteria,  but 
there  is  little  tendency  for  the  daughter-cells  to  adhere 
together  or  to  form  chains.  In  cultures  they  are  com- 
monly massed  together,  by  a  gelatinous  material,  in  the 
form  of  zoogloea.  They  do  not  form  spores,  and  are 
probably  not  motile,  though  Winogradsky  believes 
he  has  occasionally  detected  them  in  active  motion. 
As  has  been  stated,  they  do  not  grow  upon  ordinary 
nutrient  media,  and  cannot,  therefore,  be  isolated  by 
the  means  commonly  employed  to  separate  different 
species  of  bacteria.  The  most  astonishing  property  of 
this  organism  is  its  ability  to  grow  and  perform  its 
specific  fermentative  function  in  solutions  devoid  of 
organic  matter.  It  is  believed  to  be  able  to  obtain  its 
necessary  carbon  from  carbonic  acid.  For  its  isolation 
and  cultivation  Winogradsky  recommends  the  following 
solution : 

Ammonium  sulphate 1  gramme. 

Potassium  phosphate 1        " 

Pure  water 1000  c.c. 

To  each  flask  containing  100  c.c.  of  this  fluid  is  added 
from  0.5  to  1  gramme  of  basic  magnesium  carbonate 
suspended  in  a  little  distilled  water  and  sterilized  by 
boiling.  One  of  the  flasks  is  then  to  be  inoculated  with 
a  minute  portion  of  the  soil  under  investigation,  and 
after  four  or  five  days  a  small  portion  is  to  be  with- 
drawn, by  means  of  a  capillary  pipette,  from  over  the 
surface  of  the  layer  of  magnesium  carbonate  and  trans- 
ferred to  a  second  flask,  and  similarly  after  four  or  five 
days  from  this  to  a  third  flask,  and  so  on.  As  this 
medium  does  not  offer  conditions  favorable  to  the 


THE  NITRIFYING  BACTERIA.  467 

growth  of  bacteria  requiring  organic  matter  for  their 
development,  those  that  were  originally  introduced  with 
the  soil  quickly  disappear,  and  ultimately  only  the  nitri- 
fying organisms  remain.  These  are  seen  as  an  almost 
transparent  film  attached  to  the  clumps  and  granules  of 
magnesium  carbonate  on  the  bottom  of  the  flask. 

For  their  cultivation  upon  a  solid  medium  Winograd- 
sky  employs  a  mineral  gelatin,  the  gelatinizing  principle 
of  which  is  silicic  acid.  A  solution  of  from  3  to  4  per 
cent,  of  silicic  acid  in  distilled  water,  and  having  a  spe- 
cific gravity  of  1.02,  remains  fluid  and  can  be  preserved 
in  flasks  in  this  condition.  (Kiihne.)  Gelatinization  oc- 
curs after  the  addition  of  certain  salts  to  such  a  solution, 
and  will  be  more  or  less  complete  according  to  the  pro- 
portion of  salts  added.  The  salts  that  have  given  the 
best  results  and  the  method  of  mixing  them  are  as  fol- 
lows : 

f  Ammonium  sulphate 0.4  gramme. 

a  •<  Magnesium  sulphate 0.05  " 

( Calcium  chloride trace. 

(  Potassium  phosphate .t  ...  0.1  gramme. 

6  \  Sodium  carbonate 0.6  to  0.9  " 

(  Distilled  water 100  c.c. 

The  sulphates  and  chloride  (a)  are  mixed  in  50  c.c. 
of  the  distilled  water,  and  the  phosphate  and  carbonate 
(b)  in  the  remaining  50  c.c.,  in  separate  flasks. 

Each  flask  is  then  sterilized  with  its  contents,  which 
after  cooling  are  mixed ;  the  mixture  representing  the 
solution  of  mineral  salts  is  to  be  added  to  the  silicic 
acid,  little  by  little,  until  the  proper  degree  of  consist- 
ency is  obtained  (that  of  ordinary  nutrient  gelatin). 
This  part  of  the  process  is  best  conducted  in  a  culture- 
dish.  If  it  is  desired  to  separate  the  colonies,  as  in  an 
ordinary  plate,  the  inoculation  and  mixing  of  the  mate- 


468  BACTERIOLOGY. 

rial  introduced  must  be  done  before  gelatinization  is 
complete ;  if  the  material  is  to  be  distributed  over  only 
the  surface  of  the  medium,  then  the  mixture  must  first 
be  allowed  to  solidify. 

By  the  use  of  the  silicate-gelatin  Winogradsky  has 
isolated  from  the  gelatinous  film  in  the  bottom  of  fluids 
undergoing  nitrification  a  bacillus  which  he  believes  to  be 
associated  with  the  nitro-monas  in  the  nitrifying  process. 

Our  knowledge  of  these  organisms  is  as  yet  too  in- 
complete to  permit  of  a  complete  description.  What 
has  been  said  will  serve  to  indicate  the  direction  in 
which  further  studies  of  the  subject  should  be  prose- 
cuted. (For  further  details,  the  reader  is  referred  to  the 
original  contributions  and  to  current  literature  on  the 
subject.1) 

In  addition  to  the  bacteria  concerned  in  decomposition 
and  nitrification  there  are  occasionally  present  in  the 
soil  micro-organisms  possessing  disease-producing  prop- 
erties. Conspicuous  among  these  may  be  mentioned 
the  bacillus  of  malignant  oedema  (vibrion  septique  of 
the  French),  the  bacillus  of  tetanus,  and  the  bacillus 
of  symptomatic  anthrax  (Rausehbrand  (Ger.) ;  charbon 
symptomatique  (Fr.)).  It  is  sometimes  due  to  the 
presence  of  one  or  the  other  of  these  organisms  that 
wounds  to  which  soil  has  had  access  (crushed  wounds 
from  the  wheels  of  cars  or  wagons,  wounds  received  in 
agricultural  work,  etc.)  are  followed  by  such  grave  con- 
stitutional disturbances. 

1  Winogradsky:  Ann.  de  Flnst.  Pasteur,  1890,  tome  iv. ;  1891,  tome  v. 

Jordan  and  Eichards:  Eeport  of  State  Board  of  Health  of  Massa- 
chusetts, "Purification  of  Sewage. and  Water,"  1890,  vol.  ii.  p.  864. 

Frankland,  G.  C.  and  P.  F. :  Proceedings  of  Eoyal  Society,  London, 
1890,  xlvii. 


THE  BACILLUS  OF  TETANUS.  469 

.      THE    BACILLUS   OF   TETANUS. 

In  1884  Nicolaier  produced  tetanus  in  mice  and  rab- 
bits by  the  subcutaneous  inoculation  of  particles  of 
garden-earth,  and  demonstrated  that  the  pus  produced 
at  the  point  of  inoculation  was  capable  of  reproducing 
the  disease  in  other  mice  and  rabbits.  He  did  not  suc- 
ceed in  isolating  the  organism  in  pure  culture.  In  1884 
Carle  and  Rattone,  and  in  1886  Rosenbach,  demon- 
strated the  infectious  nature  of  tetanus  as  it  occurs  in 
man  by  producing  the  disease  in  animals  by  inoculating 
them  with  secretions  from  the  wounds  of  individuals 
affected  with  the  disease.  In  1889  Kitasato  obtained 
the  bacillus  of  tetanus  in  pure  culture,  and  described 
his  method  of  obtaining  it  and  its  biological  peculiarities 
as  follows : 

METHOD  OF  OBTAINING  IT. — Inoculate  several  mice 
subcutaneously  with  secretions  from  the  wound  of  a 
case  of  typical  tetanus.  This  material  usually  contains 
not  only  tetanus  bacilli,  but  other  organisms  as  well,  so 
that  at  autopsy,  if  tetanus  results,  there  may  be  more  or 
less  suppuration  at  the  seat  of  inoculation  in  the  mice. 
In  order  to  separate  the  tetanus  bacillus  from  the  others 
that  are  present  the  pus  is  smeared  upon  the  surface 
of  several  slanted  blood-serum  or  agar-agar  tubes  and 
placed  at  37°  to  38°  C.  After  twenty-four  hours  all 
the  organisms  will  have  developed,  and  microscopic 
examination  will  usually  reveal  the  presence  of  a  few 
tetanus  bacilli,  recognizable  by  their  shape,  viz.,  that  of 
a  small  pin,  with  a  spore  representing  the  head.  After 
forty-eight  hours  at  38°  C.  the  culture  is  subjected  to  a 
temperature  of  80°  C.  in  a  -water-bath  for  from  three- 
quarters  to  one  hour.  At  the  end  of  this  time  series  of 


470  BACTERIOLOGY. 

plates  or  Esmarch  tubes  of  slightly  alkaline  gelatin  are 
made  with  very  small  amounts  of  the  culture  and  kept 
in  an  atmosphere  of  hydrogen  (see  page  210).  They 
are  then  kept  at  from  18°  to  20°  C.,  and  at  the  end 
of  about  a  week  the  tetanus  bacillus  begins  to  appear 
in  the  form  of  colonies.  After  about  ten  days  the 
colonies  should  not  only  be  examined  microscopically, 
but  each  colony  that  has  developed  in  the  hydrogen 
atmosphere  should  be  obtained  in  pure  culture  and 
again  grown  under  the  same  conditions.  The  colonies 
that  grow  only  without  oxygen,  and  which  are  com- 
posed of  the  pin-shaped  organisms,  must  be  tested  upon 
mice.  If  they  represent  growths  of  the  tetanus  bacillus, 
the  typical  clinical  manifestations  of  the  disease  will  be 
produced  in  these  animals. 

In  obtaining  the  organism  from  the  soil  much  diffi- 
culty is  experienced.  Here  are  encountered  a  number 
of  spore-bearing  organisms  that  are  facultative  in 
their  relation  to  oxygen,  and  are  therefore  very  difficult 
to  eliminate ;  and  there  is,  moreover,  one  in  particular 
that,  like  the  tetanus  bacillus,  forms  a  polar  spore. 
This  spore  is,  however,  less  round  and  much  more  oval 
than  that  of  the  tetanus  bacillus,  and  gives  to  the 
organism  containing  it  more  the  shape  of  a  javelin  (or 
clostridium,  properly  speaking)  than  that  of  a  pin,  the 
characteristic  shape  of  the  spore-bearing  tetanus  organ- 
ism. It  is  non-pathogenic,  and  grows  both  with  and 
without  oxygen,  and  should,  consequently,  not  be  mis- 
taken for  the  latter  bacillus.  It  must  also  be  borne  in 
mind  that  there  are  occasionally  present  in  the  soil  still 
other  bacilli  which  form  polar  spores,  and  which,  when 
in  this  stage,  are  almost  identical  in  appearance  with 
the  tetanus  bacillus ;  but  they  will  usually  be  found  to 


THE  BACILLUS  OF  TETANUS. 


471 


differ  from  it  in  their  relation  to  oxygen,  and  they  are 
also  without  disease-producing  properties. 

MORPHOLOGY. — It  is  a  slender  rod  with  rounded  ends. 
It  may  appear  as  single  rods,  or,  in  cultures,  as  long 
threads.  It  is  motile,  though  not  actively  so.  The 
motility  is  rendered  somewhat  more  conspicuous  by 
examining  the  organism  upon  a  warm  stage. 

FIG.  94. 


Bacillus  tetani.   A.  Vegetative  stage.   B.  Spore-stage,  showing  pin-shapes. 


At  the  temperature  of  the  body  it  rapidly  forms 
spores.  These  are  round,  thicker  than  the  cell,  and 
usually  occupy  one  of  its  poles,  giving  to  the  rod  the 
appearance  of  a  small  pin.  (Fig-  94.)  When  in  the 
spore-stage  it  is  not  motile. 

It  is  stained  by  the  ordinary  aniline  staining-reagents. 
It  retains  the  color  when  stained  by  Gram's  method. 

CULTURAL  PECULIARITIES. — It  is  an  obligate  anae- 
robe, and  cannot  be  brought  to  development  under 
access  of  oxygen.  It  thrives  in  an  atmosphere  of 
pure  hydrogen,  but  not  in  one  of  carbonic  acid. 


472 


BACTERIOLOGY. 


FIG.  95. 


It  grows  in  ordinary  nutrient  gelatin  and  agar-agar 
of  a  slightly  alkaline  reaction.  Gelatin  is  slowly  lique- 
fied, with  the  coincident  production  of  a  small  amount 
of  gas.  Neither  agar-agar  nor 
blood-serum  is  liquefied  by  its 
growth. 

The  addition  to  the  media  of 
from  1.5  to  2  per  cent,  of  glucose, 
0.1  per  cent,  of  indigo-sodium 
sulphate,  or  5  per  cent,  by  volume 
of  blue  litmus  tincture  favors  its 
growth. 

It  grows  well  in  alkaline  bouil- 
lon under  an  atmosphere  of  hy- 
drogen. 

Under  artificial  conditions  it 
may  be  cultivated  through  nu- 
merous generations  without  loss 
of  virulence. 

APPEARANCE  OF  THE  COLO- 
NIES.— Colonies  of  bacillus  tetani 
on  gelatin  under  an  atmosphere 
of  hydrogen  have,  in  their  early 
stages  somewhat  the  appearance 
of  the  colonies  of  the  common 
bacillus  subtilis  in  their  earliest 
stages,  viz.,  they  have  a  dense, 
felt-like  centre  surrounded  by  a 
fringe  of  delicate  radii.  The 
liquefaction  is  so  slow  that  the 
appearance  is  retained  for  a  rela- 
tively long  time,  but  eventually 
becomes  altered.  In  very  old 


Colonies  of  the  tetanus 
bacillus  four  days  old.made 
by  distributing  the  organ- 
isms through  a  tube  nearly 
filled  with  glucose-gel- 
atin. Cultivation  in  an  at- 
mosphere of  hydrogen. 
(From  FRANKEL  and 
PFEIFFER.) 


THE  BACILLUS  OF  TETANUS.  473 

colonies  the  entire  mass  is  made  up  of  a  number  of 
distinct  threads  that  give  it  the  appearance  of  a  com- 
mon mould.  (See  Fig.  95.) 

In  stab-cultures  made  in  tubes  about  three-quarters 
filled  with  gelatin  growth  begins  at  about  1.5  to  3  cm. 
below  the  surface,  and  gradually  assumes  the  appearance 
of  a  cloudy,  linear  mass,  with  prolongations  radiating 
into  the  gelatin  from  all  sides.  Liquefaction  with  coinci- 
dent gas-production  results,  and  may  reach  almost  to 
the  surface  of  the  gelatin. 

RELATION  TO  TEMPERATURE  AXD  TO  CHEMICAL 
AGENTS. — It  grows  best  under  a  temperature  of  from 
36°  to  38°  C. ;  gelatin  cultures  kept  at  from  20°  to 
25°  C.  begin  to  grow  after  three  or  four  days.  In  an 
atmosphere  of  hydrogen  at  from  18°  to  20°  C.  growth 
does  not  usually  occur  before  one  week.  No  growth 
occurs  below  14°  C.  At  the  temperature  of  the  body 
spores  are  formed  in  cultures  in  about  thirty  hours, 
whereas  in  gelatin  cultures  at  from  20°  to  25°  C.  they 
do  not  usually  appear  before  a  week,  when  the  lower 
part  of  the  gelatin  is  quite  fluid. 

Spores  of  the  tetanus  bacillus  when  dried  upon  bits 
of  thread  over  sulphuric  acid  in  the  desiccator  and  sub- 
sequently kept  exposed  to  the  air,  retain  their  vitality 
and  virulence  for  a  number  of  months.  Their  vitality 
is  not  destroyed  by  an  exposure  of  one  hour  to  80°  C. ; 
on  the  other  hand,  an  exposure  of  five  minutes  to 
100°  C.  in  the  steam  sterilizer  kills  them.  They  resist 
the  action  of  5  per  cent,  carbolic  acid  for  ten  hours,  but 
succumb  when  exposed  to  it  for  fifteen  hours.  In  the 
same  solution,  plus  0.5  per  cent,  of  hydrochloric  acid, 
they  are  no  longer  active  after  two  hours.  They  are 
killed  when  acted  upon  for  three  hours  by  corrosive 


474  BACTERIOLOGY. 

sublimate,  1  : 1000,  and  in  thirty  minutes  by  the  same 
solution  plus  0.5  per  cent,  of  hydrochloric  acid. 

ACTION  UPON  ANIMALS. — After  subcutaneous  inocu- 
lation of  mice  with  minute  portions  of  a  pure  culture 
of  this  organism  tetanus  develops  in  twenty-four  hours 
and  ends  fatally  in  from  two  to  three  days.  Rats, 
guinea-pigs,  and  rabbits  are  similarly  affected,  but  only 
by  larger  doses  than  are  required  for  mice,  the  fatal 
dose  for  a  rabbit  being  from  0.3  to  0.5  c.c.  of  a  well- 
developed  bouillon  culture.  The  period  of  incubation 
for  rats  and  guinea-pigs  is  twenty-four  to  thirty  hours, 
and  for  rabbits  from  two  to  three  days.  Pigeons  are 
but  slightly,  if  at  all,  susceptible. 

The  tetanic  convulsions  always  appear  first  in  the 
parts  nearest  the  seat  of  inoculation,  and  subsequently 
become  general. 

At  autopsies  upon  animals  that  have  succumbed  to 
inoculations  with  pure  cultures1  of  bacillus  tetani  there 
is  little  to  be  seen  by  either  macroscopic  or  micro- 
scopic examination,  and  cultures  from  the  site  of  inocu- 
lation are  usually  negative  in  so  far  as  finding  the  teta- 
nus bacillus  is  concerned.  At  the  site  of  inoculation 
there  is  usually  only  a  hypersemic  condition.  In  un- 
complicated cases  there  is  no  suppuration.  The  internal 
organs  do  not  present  any  change,  and  culture-methods 
of  examination  show  them  to  be  free  from  bacteria. 
The  death  of  the  animal  results  from  the  absorption  of 
a  soluble  poison,  either  produced  by  the  bacteria  at  the 
site  of  inoculation  or,  which  seems  more  probable,  pro- 

1  Animals  and  human  beings  that  have  become  infected  with  this 
organism  in  the  ordinary  way  commonly  present  a  condition  of  sup- 
puration at  the  site  of  infection  ;  this  is  probably  not  due,  however,  to 
the  tetanus  bacillus,  but  to  other  bacteria  that  have  also  gained  access 
to  the  wound  at  the  time  of  infection. 


THE  BACILLUS  OF  TETANUS.  475 

duced  by  the  bacteria  in  the  culture  from  which  they  are 
obtained  and  introduced  with  them  into  the  tissues  of 
the  animal  at  the  time  of  inoculation.  In  support 
of  the  latter  hypothesis  :  mice  have  been  inoculated  with 
pure  cultures  of  this  organism  ;  after  one  hour  the  point 
at  which  the  inoculation  was  made  was  excised  and  the 
tissues  cauterized  with  the  hot  iron ;  notwithstanding 
the  short  time  during  which  the  organisms  were  in 
contact  with  ths  tissues  and  the  subsequent  radical 
treatment,  the  animals  died  after  the  usual  interval 
and  witli  the  typical  symptoms  of  tetanus. 

The  poison  produced  by  the  tetanus  bacillus,  and 
to  which  the  symptoms  of  the  disease  are  due,  has 
been  isolated  and  subjected  to  detailed  study;  some 
of  its  peculiarities,  as  given  by  Kitasato,  are  as  fol- 
lows : ' 

"When  cultures  of  this  organism  are  robbed  of  their 
bacteria  by  filtration  through  porcelain  the  filtrate  con- 
tains the  soluble  poison,  and  is  capable,  when  injected 
into  animals,  of  causing  tetanus. 

"Inoculations  of  other  animals  with  bits  of  the 
organs  of  the  animal  dead  from  the  action  of  the  teta- 
nus poison  produce  no  result ;  but  similar  inoculations 
with  the  blood  or  with  the  serous  exudate  from  the 
pleural  cavity  always  result  in  the  appearance  of  teta- 
nus. The  poison  is,  therefore,  largely  present  in  the 
circulating  fluids. 

"  The  greatest  amount  of  poison  is  produced  by  culti- 
vation in  fresh  neutral  bouillon  of  a  very  slightly  alka- 
line reaction. 

"  The  activity  of  the  poison  is  destroyed  by  an  ex- 

1  Zeitschrift  fur  Hygiene,  1891,  Bd.  x.  S.  267. 


476  BACTERIOLOGY. 

posure  of  one  and  one-half  hours  to  55°  C. ;  of  twenty 
minutes  to  60°  C. ;  and  of  five  minutes  to  65°  C. 

"By  drying  at  the  temperature  of  the  body  under 
access  of  air  the  poison  is  destroyed ;  but  by  drying  at 
the  ordinary  temperature  of  the  room,  or  at  this  tem- 
perature in  the  desiccator  over  sulphuric  acid,  it  is  not 
destroyed. 

"Diffuse  daylight  diminishes  the  intensity  of  the 
poison.  Its  intensity  is  preserved  for  a  much  longer 
time  when  kept  in  the  dark. 

"Direct  sunlight  robs  it  of  its  poisonous  properties 
in  from  fifteen  to  eighteen  hours. 

"  Its  activity  is  not  diminished  by  diluting  a  fixed 
amount  with  water  or  nutrient  bouillon. 

"  Mineral  acids  and  strong  alkalies  lessen  its  intensity." 

The  chemical  nature  of  this  poison  is  not  positively 
known,  but  according  to  the  observations  of  Brieger 
and  Cohn l  it  is  not  to  be  classed  with  the  albumins 
in  the  sense  in  which  the  word  is  commonly  used. 
When  obtained  in  a  pure,  concentrated  form  its  toxic 
properties  are  seen  to  be  altered  by  acids,  by  alkalies, 
by  sulphuretted  hydrogen,  and  by  temperatures  above 
70°  C.  Even  when  carefully  protected  from  light, 
moisture,  and  air  it  gradually  becomes  diminished  in 
strength.  When  freshly  prepared  by  the  methods  of 
the  authors  just  cited  its  potency  is  almost  incredible, 
0.00005  milligramme  being  sufficient  to  cause  fatal 
tetanus  in  a  mouse  weighing  fifteen  grammes. 

THE    BACILLUS    OF    MALIGNANT   (EDEMA. 

The  bacillus  of  malignant  oedema,  also  known  as 
vibrion  septique,  is  another  pathogenic  form  almost 

1  Zeit.  fur  Hygiene  und  Infektionskrankheiten,  1893,  Band.  xv.  S.  1. 


THE  BACILLUS  OF  MALIGNANT  (EDEMA.     477 

everywhere  present  in  the  soil.  In  certain  respects  it 
is  a  little  like  the  bacillus  of  anthrax,  and  was  at  one 
time  confounded  with  it ;  but  it  differs  in  the  marked 
peculiarity  of  being  a  strict  anaerobe.  It  was  first 
observed  by  Pasteur,  but  it  was  not  until  later  that 
Koch,  Laborious,  Kitt,  and  others  described  its  pecu- 
liarities in  detail.  It  can  usually  be  obtained  by 

FIG.  96. 


a  "a 


B 


Bacillus  oedematis  maligni.  A.  (Edema-fluid,  from  site  of  inoculation  of 
guinea-pig,  showing  long  and  short  threads.  B.  Spore-formation,  from 
culture. 

inserting  under  the  skin  of  rabbits  or  guinea-pigs 
small  portions  of  garden-earth,  street-dust,  or  decom- 
posing organic  substances.  There  results  a  widespread 


478 


BACTERIOLOGY. 


oedema,  with  more  or  less  gas-production  in  the  tissues. 

In  the  cedematous  fluid  about  the  site  of  inoculation 
the  organism  under  consideration  may 
be  detected.  (Fig.  96,  A.) 

It  is  a  rod  about  3  to  3.5  fj.  long  and 
from  1  to  1.1  fj.  thick — i.  e.,  it  is  about 
as  long  as  bacillus  anthracis }  but  is  a 
trifle  more  slender.  It  is  usually  found 
in  pairs,  joined  end  to  end,  but  may  oc- 
cur as  longer  threads ;  particularly  is 
this  the  case  in  cultures.  When  in 
pairs  the  ends  that  approximate  are 
squarely  cut,  while  the  distal  ex- 
tremities are  rounded.  When  occur- 
ring singly  both  ends  are  rounded. 
(How  does  it  differ  in  this  respect 
from  bacillus  anthracis  f)  It  is  slowly 
motile,  and  its  flagella  are  located 
both  at  the  ends  and  along  the  sides 
of  the  rod.  It  forms  spores  that  are 
usually  located  in  or  near  the  middle 
of  the  cells,  causing  frequently  a 
swelling  at  the  points  at  which  they 
are  located  and  giving  to  the  cell  a 
more  or  less  oval,  spindle,  or  lozenge 
shape.  (Fig;.  96,  B.) 

Colonies  of  the  ba-  5          ' 

ciiius  of  malignant        It  is  an  obligate  anaerobe,  growing 


FRANKEL  and  PFEIF-     with  aCC6SS  of  Oxygen.       It  gl'OWS  well 

in    an   atmosphere   of  hydrogen.      It 
causes  liquefaction  of  gelatin. 

In  tubes  containing  about  20  to  30  c.c.  of  gelatin 
that  has  been  liquefied,  inoculated  with  a  small  amount 


THE  BACILLUS  OF  MALIGNANT  (EDEMA.     479 

of  the  culture,  and  then  rapidly  solidified  in  ice-water, 
growth  appears  in  the  form  of  isolated  colonies  at  or  near 
the  bottom  of  the  tube  in  from  two  to  three  days  at  20°  C. 
These  colonies,  when  of  from  0.5  to  1  mm.  in  diameter, 
appear  as  spheres  filled  with  clear  liquid,  and  are  diffi- 
cult, for  this  reason,  to  detect.  (Fig.  97.)  As  they  grad- 
ually increase  in  size  the  contents  of  the  spheres  become 
cloudy  and  marked  by  fine  radiating  stripes,  easily 
to  be  detected  with  the  aid  of  a  small  hand-lens.  In 
deep  stab-cultures  in  agar-agar  and  in  gelatin  develop- 
ment occurs  only  along  the  track  of  puncture,  at  a  dis- 
tance below  the  surface.  Growth  is  frequently  accom- 
panied by  the  production  of  gas-bubbles. 

It  causes  rapid  liquefaction  of  blood-serum,  with 
production  of  gas-bubbles,  and  in  two  or  three  days 
the  entire  medium  may  have  become  converted  into  a 
yellowish,  semi-fluid  mass. 

The  most  satisfactory  results  in  the  study  of  the 
colonies  are  obtained  by  the  use  of  plates  of  nutrient 
agar-agar  kept  in  a  chamber  in  which  all  oxygen  has 
been  replaced  by  hydrogen.  The  colonies  appear  as 
dull  whitish  points,  irregular  in  outline,  and  when 
viewed  with  a  low-power  lens  are  seen  to  be  marked 
by  a  network  of  branching  and  interlacing  lines  that 
radiate  in  an  irregular  way  from  the  centre  toward  the 
periphery. 

It  grows  well  at  the  ordinary  temperature  of  the 
room,  but  reaches  its  highest  development  at  the  tem- 
perature of  the  body. 

It  stains  readily  with  the  ordinary  aniline  dyes.  It 
does  not  stain  by  Gram's  method. 

PATHOGEXESIS. — The  animals  known  to  be  suscepti- 
ble to  inoculation  with  this  organism  are  man,  horses, 


480  BA  CTERIOLOG  Y. 

calves,  dogs,  goats,  sheep,  pigs,  chickens,  pigeons,  rab- 
bits, guinea-pigs,  and  mice.  Cases  are  recorded  in 
which  men  and  horses  have  developed  the  disease  after 
injuries,  doubtless  due  to  the  introduction  into  the 
wound,  at  the  time,  of  soil  or  dust  containing  the 
organism. 

If  one  introduce  into  a  pocket  beneath  the  skin  of  a 
susceptible  animal  about  as  much  garden-earth  as  can 
be  held  upon  the  point  of  a  penknife,  the  animal  fre- 
quently dies  in  from  twenty-four  to  forty-eight  hours. 
The  most  conspicuous  result  found  at  autopsy  is  a  wide- 
spread oedema  at  and  about  the  site  of  inoculation.  The 
oedematous  fluid  is  in  some  places  clear,  while  at  others  it 
may  be  stained  with  blood ;  it  is  usually  rich  in  bacilli 
(Fig.  94,  A)  and  contains  gas-bubbles.  Of  the  internal 
organs  only  the  spleen  shows  much  change.  It  is  large, 
dark  in  color,  and  contains  numerous  bacilli.  If  the 
autopsy  be  made  immediately  after  death,  bacilli  are 
rarely  found  in  the  blood  of  the  heart;  but  if  de- 
ferred for  several  hours,  the  organisms  will  be  found 
in  this  locality  also,  a  fact  that  speaks  for  their  multi- 
plication in  the  body  after  death.  At  the  moment 
of  death  they  are  present  in  varying  numbers  in  all 
the  internal  viscera  and  on  the  serous  surfaces  of  the 
organs. 

Of  all  animals  mice  are  probably  the  most  susceptible 
to  the  action  of  this  organism,  and  it  is  not  rare  to  find 
it  in  the  heart's  blood,  even  immediately  after  death. 
They  die,  as  a  result  of  these  inoculations,  in  from  six- 
teen to  twenty  hours. 

When  a  pure  culture  is  used  for  inoculation  a  rela- 
tively large  amount  must  be  employed,  and  this  should  be 
introduced  into  a  deep  pocket  in  the  subcutaneous  tissues 


THE  BACILLUS  OF  MALIGNANT  (EDEMA.     481 

some  distance  from  the  surface.  In  continuing  the  in- 
oculations from  animal  to  animal  small  portions  of 
organs  or  a  few  drops  of  the  oedema-fluid  should  be 
used.  The  inoculation  may  also  be  successfully  made 
by  introducing  into  a  pocket  in  the  skin  bits  of  steril- 
ized thread  or  paper  upon  which  cultures  have  been 
dried. 

The  methods  for  obtaining  the  organism  in  pure  cult- 
ure, from  the  cadaver  of  an  animal  that  has  succumbed 
to  infection  by  the  bacillus  of  malignant  oedema,  are 
in  all  essential  respects  the  same  as  those  given 
for  obtaining  cultures  from  tissues  in  general ;  but  it 
must  be  remembered  that  the  organism  is  a  strict  anae- 
robe, and  will  not  grow  under  the  influence  of  oxygen. 
(See  methods  of  cultivating  anaerobic  species.) 

In  certain  superficial  respects  this  bacillus  suggests 
bacillus  anthracis,  but  differs  from  it  in  so  many  im- 
portant details  that  there  is  no  excuse  for  confounding 
the  two. 

NOTE. — From  what  has  been  said  of  this  organism, 
what  are  the  most  important  differential  points  between 
it  and  bacillus  anthracis  £  Inoculate  several  mice  with 
small  portions  of  garden-earth  and  street-dust.  Isolate 
the  organism  that  agrees  most  nearly  with  the  descrip- 
tion here  given  for  the  bacillus  of  malignant  oedema. 
Compare  its  morphological,  biological,  and  pathogenic 
peculiarities  with  those  of  bacillus  anthracis  under  simi- 
lar circumstances ;  especially  its  action  on  animals  and 
its  appearance  in  the  tissues  and  fluids. 

Still  another  pathogenic  organism  that  may  be  present 
in  the  soil  is 

31 


482  BACTERIOLOGY. 

THE  BACILLUS  OF  SYMPTOMATIC  ANTHRAX  ;  BACTERIE 
DU  CHARBON  SYMPTOMATIQUE  (FR.)  ;  BACILLUS 
DES  RAUSCHBRAND  (GER.). 

It  is  the  organism  concerned  in  the  production 
of  the  disease  of  young  cattle  and  sheep  commonly 
known  as  "  black  leg/'  "  quarter  evil,"  and  "  quarter 
ill/'  a  disease  that  prevails  in  certain  localities  dur- 
ing the  warm  months,  and  which  is  characterized  by 
a  peculiar  ernphysematous  swelling  of  the  muscular 
and  subcutaneous  cellular  tissues  over  the  quarters. 
The  muscles  and  cellular  tissues  at  the  points  af- 
fected are  seen  on  section  to  be  saturated  with  bloody 
serum,  and  the  muscles  particularly  are  of  a  dark, 
almost  black  color.  In  these  areas,  in  the  bloody  trans- 
udates  of  the  serous  cavities,  in  the  bile,  and,  after 
death,  in  the  internal  organs,  the  organism  to  be  de- 
scribed can  always  be  detected.  It  is  manifest  from 
this  that  the  soil  of  localities  over  which  infected  herds 
are  grazing  may  readily  become  contaminated  through 
a  variety  of  channels,  and  thus  serve  as  a  source  of 
further  dissemination  of  the  disease. 

The  organism  was  first  observed  by  Feser,  and  subse- 
quently by  Bollinger  and  others.  The  most  complete 
description  of  its  morphological  and  biological  peculi- 
arities is  that  of  Kitasato.1  The  following  is  from 
Kitasato's  contributions :  it  is  an  actively  motile  rod 
about  3  to  5  //  long  by  0.5  to  0.6  //  thick.  It  has 
rounded  ends,  and,  as  a  rule,  is  seen  singly,  though 
now  and  then  pairs  joined  end  to  end  may  occur.  It 
has  no  tendency  to  form  very  long  threads.  (Fig.  98,  A.) 

It  forms  spores,  and  when  in  this  stage  is  seen  to  be 

iZeitschrift  fur  Hygiene,  Bd.  vi.  S.  105  ;  Bd.  viii.  S.  55. 


THE  BACILLUS  OF  SYMPTOMATIC  ANTHRAX.  483 

slightly  swollen  at  or  near  one  of  its  poles,  the  location 
in  which  the  spore  usually  appears.  (Fig.  98,  B.)  It  is 
markedly  prone  to  undergo  degenerative  changes,  and 
involution-forms  are  commonly  seen,  not  only  in  fresh 
cultures,  but  in  the  tissues  of  aifected  animals  as  well. 
Though  actively  motile  when  in  the  vegetative  stage 
it,  like  all  other  motile  spore-forming  bacilli,  loses  this 

FIG.  98. 


Bacillus  of  symptomatic  anthrax.    A.  Vegetative  stage— gelatin  culture. 
B.  Spore-forms— agar-agar  culture. 

property  and  becomes  motionless  when  spores  are  form- 
ing. 

It  is  strictly  anaerobic  and  cannot  be  cultivated  in  an 
atmosphere  in  which  oxygen  is  present.  It  grows  best 
under  hydrogen,  and  does  not  grow  under  carbonic  acid. 

The  media  most  favorable  to  its  growth  are  those  con- 
taining glucose  (1.5  to  2  per  cent.),  glycerin  (4  to  5  per 
cent.),  or  some  other  reducing-body,  such  as  indigo-sodium 
sulphate,  sodium  formate,  etc. 

When  cultivated  upon  gelatin  plates  in  an  atmosphere 


484 


BACTERIOLOGY. 


FIG. 


of  hydrogen  the  colonies  appear  as  irregular,  slightly  lob u- 
lated  masses.  After  a  short  time  liquefaction  of  the  gela- 
tin occurs  and  the  colony  presents  a  dark, 
dense,  lobulated  and  broken  centre,  sur- 
rounded by  a  much  more  delicate,  fringe- 
like  zone. 

When  distributed  through  a  deep 
layer  of  liquefied  gelatin  that  is  sub- 
sequently solidified  colonies  develop  at 
only  the  lower  portions  of  the  tube. 
The  single  colonies  appear  as  discrete 
globules  that  cause  rapid  liquefaction 
of  the  gelatin,  and  ultimately,  coalesce 
into  irregular,  lobulated,  liquid  areas. 
In  some  of  the  larger  colonies  an  ill- 
defined,  concentric  arrangement  of  al- 
ternate clear  and  cloudy  zones  can  be 
made  out.  (Fig.  99.) 

In  deep  stab-cultures  in  gelatin  growth 
begins  after  about  two  or  three  days  at 
20°  to  25°  C.  It  begins  usually  at  about 
one  or  two  centimetres  below  the  sur- 
face, and  causes  slow  liquefaction  at  and 
around  the  track  of  its  development. 
During  its  growth  gas-bubbles  are  pro- 
duced. 

In    deep    stab-cultures   in   agar-agar 
at  37°  to  38°  C.  growth  begins  in  from 
twenty-four  to  forty-eight  hours,  also  at 
about  one  or  two  centimetres  below  the 
surface,  and  is  accompanied  by  the  production  of  gas- 
bubbles.     There  is  produced  at  the  same  time  a  peculiar, 
penetrating  odor  somewhat  suggestive  of  that  of  rancid 


Colonies  of  the 
bacillus  of  symp- 
tomatic anthrax, 
in  deep  gelatin 
culture.  (After 
FRANKEL  and 
PFEIFFER.) 


THE  BACILLUS  OF  SYMPTOMATIC  ANTHRAX.  485 

butter.  Under  these  conditions  spores  are  formed  after 
about  thirty  hours. 

It  grows  well  in  bouillon  of  very  slightly  acid  reac- 
tion under  hydrogen,  but  does  not  retain  its  virulence 
for  so  long  a  time  as  when  cultivated  upon  solid  media. 
In  this  medium  it  develops  in  the  form  of  white  flocculi 
that  sink  ultimately  to  the  bottom  of  the  glass  and  leave 
the  supernatant  fluid  quite  clear.  If  the  vessel  be  now 
gently  shaken,  these  delicate  flakes  are  distributed  homo- 
geneously through  it.  In  bouillon  cultures  there  is 
often  seen  a  delicate  ring  of  gas-bubbles  round  the 
point  of  contact  of  the  tube  and  the  surface  of  the 
bouillon.  There  is  produced  also  a  peculiar,  penetrat- 
ing, sour  or  rancid  odor. 

It  grows  best  at  the  body-temperature — i.  e.,  from  37° 
to  38°  C. — but  can  also  be  brought  to  development  at 
from  16°  to  18°  C.  Below  14°  C.  no  growth  is  seen. 
Spore-formation  appears  much  sooner  at  the  higher  than 
at  the  lower  temperatures.  When  its  spores  are  dried 
upon  bits  of  thread  in  the  desiccator  over  sulphuric  acid, 
and  then  kept  under  ordinary  conditions,  they  retain 
their  vitality  and  virulence  for  many  months.  Sim- 
ilarly, bits  of  flesh  from  the  affected  areas  of  animals 
dead  of  this  disease,  when  completely  dried,  are  seen  to 
retain  for  a  long  time  the  power  of  reproducing  the 
disease.  The  spores  are  tolerably  resistant  to  the  in- 
fluence of  heat :  when  subjected  to  a  temperature  of 
80°  C.  for  one  hour  their  virulence  is  not  affected,  but 
an  exposure  to  100°  C.  for  five  minutes  destroys 
them.  They  are  also  seen  to  be  somewhat  resistant  to 
the  action  of  chemicals :  when  exposed  to  5  per  cent, 
carbolic  acid  they  retain  their  disease-producing  prop- 
erties for  about  ten  hours,  whereas  the  vegetative  forms 


486  BACTERIOLOGY. 

are  destroyed  in  from  three  to  five  minutes  ;  in  corro- 
sive sublimate  solution  of  the  strength  of  1  : 1000  the 
spores  are  killed  in  two  hours. 

When  gelatin  cultures  are  examined  microscopically 
the  organisms  are  usually  seen  as  single  rods  with 
rounded  ends.  When  cultivated  in  agar-agar  at  a 
higher  temperature  spores  are  formed  after  a  short 
time;  the  spores  are  oval,  slightly  flattened  on  their 
sides,  thicker  than  the  bacilli,  and,  as  stated,  frequently 
occupy  a  position  inclining  to  one  of  the  poles  of  the 
bacillus,  though  they  are  as  often  seen  in  the  middle. 

Bacilli  containing  spores  are  usually  clubbed  or  spin- 
dle shape. 

This  bacillus  stains  readily  with  the  ordinary  aniline 
dyes.  It  is  decolorized  by  Gram's  method.  Its  spores 
may  be  stained  by  the  methods  usually  employed  in 
spore-staining. 

PATHOGENESIS. — When  susceptible  animals,  especi- 
ally guinea-pigs,  are  inoculated  in  the  deeper  subcutane- 
ous cellular  tissues  with  pure  cultures  of  this  organism, 
or  with  bits  of  tissue  from  the  affected  area  of  another 
animal  dead  of  the  disease,  death  ensues  in  from  one  to 
two  days.  It  is  preceded  by  rise  of  temperature,  loss 
of  appetite,  and  general  indisposition.  The  site  of 
inoculation  is  swollen  and  painful,  and  drops  of  bloody 
serum  may  sometimes  be  seen  exuding  from  it.  At 
autopsy  the  subcutaneous  cellular  tissues  and  under- 
lying muscles  present  a  condition  of  emphysema  and 
extreme  oedema.  The  oedematous  fluid  is  often  blood- 
stained and  the  muscles  are  of  a  blackish  or  blackish  - 
brown  color.  The  lymphatic  glands  are  markedly 
hypersemic.  The  internal  viscera  present  but  little 
alteration  visible  to  the  naked  eye.  In  the  blood- 


THE  BACILLUS  OF  SYMPTOMATIC  ANTHRAX.  487 

stained  serous  fluid  about  the  point  of  inoculation  short 
bacilli  are  present  in  large  numbers.  These  often  pre- 
sent slight  swellings  at  the  middle  or  near  the  end. 
They  are  not  seen  as  threads,  but  lie  singly  in  the 
tissues.  Occasionally  two  will  be  seen  joined  end  to 
end.  If  the  autopsy  be  made  immediately  after  death, 
tin-so  organisms  may  not  be  detected  in  the  internal 
organs ;  but  if  not  made  until  after  a  few  hours,  they 
will  be  found  there  also.  In  recent  autopsies  only  vege- 
tative forms  of  the  organism  may  be  found;  but  later 
(in  from  twenty  to  twenty-four  hours)  spore-bearing  rods 
may  be  detected.  (How  does  this  compare  with  bacillus 
antkradsf)  By  successive  inoculations  of  susceptible 
animals  with  serous  fluid  from  the  site  of  inoculation 
of  the  dead  animal  the  disease  may  be  reproduced. 

Cattle,  sheep,  goats,  guinea-pigs,  and  mice  are  sus- 
ceptible to  infection  with  this  organism,  and  present  the 
conditions  above  described ;  whereas  horses,  asses,  and 
white  rats  present  only  local  swelling  at  the  site  of  inoc- 
ulation. Swine,  dogs,  cats,  rabbits,  ducks,  chickens, 
and  pigeons  are,  as  a  rule,  naturally  immune  from  the 
disease. 

Though  closely  simulating  the  bacillus  of  malignant 
oedema  in  many  of  its  peculiarities,  this  organism  can, 
nevertheless,  be  readily  distinguished  from  it.  It  is 
smaller;  it  does  not  develop  into  long  threads  in  the 
tissues;  it  is  more  actively  motile,  and  forms  spores 
more  readily  in  the  tissues  of  the  animal  than  does  the 
bacillus  of  malignant  oedema.  In  their  relation  to  ani- 
mals they  also  differ;  for  instance,  cattle,  while  con- 
spicuously susceptible  to  symptomatic  anthrax,  are  prac- 
tically immune  from  malignant  oedema  ;  and  while  swine, 
dogs,  rabbits,  chickens,  and  pigeons  are  readily  infected 


488  BACTERIOLOGY. 

with  malignant  oedema,  they  are  not,  as  a  rule,  suscepti- 
ble to  symptomatic  anthrax  Horses  are  affected  only 
locally,  and  not  seriously,  by  the  bacillus  of  symptomatic 
anthrax  ;  but  they  are  conspicuously  susceptible  to  both 
artificial  inoculation  and  natural  infection  by  the  bacil- 
lus of  malignant  oedema. 

The  distribution  of  the  two  organisms  over  the  earth's 
surface  is  also  quite  different.  The  oedema  bacillus  is 
present  in  almost  all  soils,  while  the  bacillus  of  symp- 
tomatic anthrax  appears  to  be  confined  to  certain  locali- 
ties, especially  places  over  which  infected  herds  have 
been  pastured. 

A  single  attack  of  symptomatic  anthrax,  if  not  fatal, 
affords  subsequent  protection ;  while  infection  with  the 
malignant  oedema  bacillus  appears  to  predispose  to  re- 
currence of  the  disease.  (Baumgarten.) 


CHAPTER    XXVI. 

Infection  and  immunity — The  types  of  infection ;  intimate  nature  of 
infection — Septicaemia,  toxaemia,  variations  in  infectious  processes 
— Immunity,  natural  and  acquired — The  hypotheses  that  have 
been  advanced  in  explanation  of  immunity — Conclusions. 

AN  organism  capable  of  producing  disease  we  call 
pathogenic  or  infective,  and  the  process  by  which  it  pro- 
duces disease  we  know  as  infection.  Diseases,  therefore, 
that  depend  for  their  existence  upon  the  presence  of 
bacteria  in  the  tissues  are  infectious  diseases. 

What  is  the  intimate  nature  of  this  process  we  call 
infection?  Is  it  due  to  the  mechanical  presence  of 
living  bacteria  in  the  body,  or  does  it  result  from  the 
deposition  in  the  tissues  of  substances  produced  by  these 
bacteria  that  are  either  locally  or  generally  incompat- 
ible with  life  ?  Or,  is  the  group  of  pathological  altera- 
tions and  constitutional  symptoms  seen  in  these  diseases 
the  result  of  abstraction  front  the  tissues,  by  the  bacteria 
growing  in  them,  of  substances  essential  to  their  vitality  ? 
These  are  some  of  the  more  important  of  the  questions 
that  present  themselves  in  the  course  of  analysis  of  this 
interesting  phenomenon. 

Let  us  look  into  several  typical  infectious  diseases, 
note  what  we  find,  and  see  to  what  extent  the  observa- 
tions thus  made  will  aid  us  in  formulating  an  opinion. 
We  begin  with  a  study  of  those  diseases  in  which  there 
is  a  general  infection^-i".  e.,  in  which  there  is  a  general  dis- 
tribution of  the  infective  agents  throughout  the  body. 

489 


490  BACTERIOLOGY. 

This  group  comprises  the  "  septicaemias,"  and  of  them 
the  disease  of  animals  known  as  anthrax  represents  a 
type  of  the  condition.  If  the  cadaver  of  an  animal 
dead  of  anthrax  be  examined  by  bacteriological  methods, 
there  will  be  discovered  present  in  all  the  organs 
and  tissues  an  organism,  a  bacillus,  of  definite  form 
and  biological  characteristics ;  and  if  the  organs,  and 
tissues  generally,  be  subjected  to  microscopic  examina- 
tion this  same  organism  will  be  found  always  present 
and  always  located  within  the  capillaries.  At  many 
points  it  will  be  seen  crowded  in  the  capillaries  in  such 
numbers  as  almost,  if  not  quite,  to  burst  them,  and  very 
commonly  their  lumen  for  a  considerable  extent  is  en- 
tirely occluded  by  the  growing  bacilli.  In  such  a  case 
as  this  we  might  be  tempted  to  conclude  that  death  had 
resulted  from  mechanical  interference  with  the  capillary 
circulation.  Suppose,  however,  we  subject  the  cultures 
obtained  from  this  animal  to  conditions,  either  chemical 
or  thermal,  that  are  not  particularly  favorable  to  their 
normal  development,  and  from  time  to  time  inoculate 
susceptible  animals  with  the  cultures  so  treated.  The 
result  will  be  that,  as  we  continue  to  expose  our  cultures 
to  unfavorable  surroundings,  the  period  of  time  that  is 
required  for  them  to  cause  the  death  of  animals  will, 
in  some  cases,  gradually  become  extended,  until  finally 
death  will  not  ensue  at  all  after  inoculation.  If,  as 
these  animals  die,  a  careful  record  of  the  conditions 
found  at  autopsy  be  kept  and  compared,  it  will  ulti- 
mately be  noticed  that  the  animals  that  die  at  a  longer 
time  after  inoculation  present  conditions  more  or  less 
at  variance  with  those  seen  in  the  original  animal  that 
died  more  quickly  after  having  been  inoculated.  These 
differences  usually  consist  in  a  diminution  of  the  num- 


INFECTION  AND  IMMUNITY. 

ber  of  bacilli  that  appear  upon  culture  plates  from  the 
blood  and  internal  organs,  and  in  a  lessening  in  the 
amount  of  mechanical  obstruction  offered  to  the  circu- 
lation through  plugging  of  the  capillaries  by  masses  of 
bacilli,  as  detected  by  microscopic  examination  of  sec- 
tions of  the  organs ;  indeed,  this  latter  condition  may 
often  have  almost,  if  not  quite,  disappeared.  We  see  here 
an  animal  dead  from  the  invasion  of  the  same  organism 
that  produced  death  in  the  first  animal,  but  with  little 
or  none  of  those  particular  lesions  to  which  we  were 
inclined  to  attribute  the  death  of  that  animal.  It  is 
apparent,  then,  that  this  organism  with  which  we  have 
been  working  can  destroy  the  vitality  of  an  animal  in 
a  way  other  than  by  mechanically  obstructing  its  blood- 
vessels ;  it  possesses  some  other  means  of  destroying 
life.  Possibly  its  growth  in  the  tissues  is  accompanied 
by  the  production  of  soluble  poisons,  which  when  pres- 
ent in  the  blood  are  not  compatible  with  life. 

Let  us  see  if  the  study  of  another  group  of  infections 
will  furnish  any  evidence  in  support  of  such  an  hypoth- 
esis. Introduce  into  the  subcutaneous  tissues  of  a 
guinea-pig  a  small  amount  of  a  pure  culture  of  the  bacil- 
lus of  diphtheria.  In  three  or  four  days  the  animal 
dies.  We  proceed  with  our  autopsy  in  exactly  the  same 
way  that  we  did  with  the  animals  dead  of  anthrax, 
and  are  astonished  to  find  that  the  organs,  blood,  and 
tissues  generally  are  sterile,1  in  so  far  as  the  presence  of 
the  organism  with  which  the  animal  was  inoculated  is 
concerned,  and  by  both  culture  and  microscopic  methods 
it  is  possible  to  detect  them  only  at  the  site  of  inocula- 
tion, where  they  were  deposited.  It  is  very  evident 

1  In  by  far  the  greater  number  of  cases  this  is  true  ;  but  under  par- 
ticular circumstances  there  are  exceptions. 


492  BACTERIOLOGY. 

that  we  have  here  a  condition  with  which  mechanical 
plugging  of  the  capillaries  could  have  had  no  connec- 
tion, for  there  are  no  organisms  in  the  blood  to  interfere 
with  its  circulation.  Our  hypothesis  then  with  regard 
to  the  condition  found  in  our  first  case  of  anthrax  is 
again  not  tenable.  Similarly,  if  an  animal  that  has 
died  of  tetanus  be  examined,  we  do  not  find  the  bacilli 
in  the  tissues  and  circulating  fluids  generally,  and,  in- 
deed, often  fail  to  find  them  at  the  point  of  injury. 
Plainly,  the  fatal  results  following  upon  inoculations 
with  the  diphtheria  and  the  tetanus  bacillus,  with  their 
accompanying  tissue-changes,  occur  from  the  presence 
of  a  something  that  cannot  be  detected  by  either  cult- 
ural or  microscopic  methods,  and  this  something  can  be 
only  a  soluble  substance  that  is  produced  by  the  growing 
bacteria  at  the  site  of  inoculation,  gains  access  to  the 
circulation,  and  through  this  channel  causes  death,  for 
it  is  scarcely  to  be  imagined  that  the  insignificant  wound 
made  in  the  course  of  inoculation  could  per  se  have  had 
this  effect.  In  other  words,  these  latter  animals  have 
died  from  what  is  called  toxaemia  (poison  in  the  blood), 
a  condition  distinctly  different  from  septiecemia,  as  seen 
in  our  first  animal  dead  of  anthrax. 

There  are,  again,  other  infectious  diseases,  many  of 
which  are  known  to  present  variations  from  what  might 
be  considered  a  typical  course,  that  may  still  further 
serve  to  support  the  view  that  infection  is  a  process  in 
which  the  mechanical  effect  of  organisms  in  the  circu- 
lating fluids  is  of  little  consequence.  Conspicuous 
among  these  are  the  infections  that  follow  upon  the 
introduction  into  the  tissues  of  susceptible  animals  of 
cultures  of  microeoccus  lanceolatiis  (pneumococcus),  of 
the  bacillus  of  chicken  cholera,  and  of  the  organisms  con- 


INFECTION  AND  IMMUNITY.  493 

cerned  in  the  production  of  the  so-called  "  hemorrhagi'5 
septicaemias."  When  running  their  normal  course  the 
specific  organisms  of  these  diseases  cause  typical  septi- 
caemias in  susceptible  animals  ;  but  often,  from  causes  not 
entirely  clear,  the  animals  die  with  only  local  lesions,  or 
with  but  very  few  organisms  in  the  internal  viscera.  We 
see  here  conditions  analogous  to  those  observed  in  the 
two  experiments  with  anthrax,  viz.,  we  find  a  group  of 
diseases  that  are  properly  classed  as  septicaemias,  be- 
cause of  the  usual  general  invasion  of  the  body  by  the 
organisms  concerned  in  their  production,  but  which 
frequently  assume  a  purely  local  character — in  both 
instances  proving  fatal  to  the  animal  infected.  From 
what  we  have  seen  it  is  manifestly  probable  that, 
whether  these  diseases  be  designated  as  septicaemias  or 
as  toxaemias,  death  is  produced  in  all  instances  by 
poisonous  substances  that  are  generated  by  the  infecting 
bacteria.  In  the  case  of  typical  anthrax,  and  other 
varieties  of  septicaemia,  the  production  of  this  poison 
is  associated  with  the  general  dissemination  of  the 
organisms  throughout  the  body ;  while  in  those  infec- 
tions often  referred  to  as  toxaemias,  of  which  diphtheria 
may  be  taken  as  a  type,  the  poison  is  produced  by  the 
organisms  that  remain  localized  at  the  site  of  invasion, 
and  is  thence  disseminated  throughout  the  body  by  the 
circulating  fluids.  Infection  thus  far,  then,  appears  to 
be  a  chemical  process. 

Through  special  investigations  that  have  been  made 
upon  the  products  of  growth  of  certain  pathogenic  bac- 
teria this  opinion  receives  further  confirmation ;  it  has 
been  found  possible  by  the  use  of  appropriate  methods 
to  isolate  from  among  the  mass  of  material  in  which 
certain  of  these  organisms  have  been  artificially  culti- 


494  BACTERIOLOGY. 

vated  substances  which,  when  separated  from  the 
bacteria  by  which  they  were  produced,  possess  the 
power  of  causing  in  animals  all  the  constitutional 
symptoms  and  pathological  tissue-changes  that  occur 
in  the  course  of  infection  by  the  organisms  themselves. 
In  some  instances  these  poisons — toxins,1  as  they  are 
collectively  called — appear  to  be  the  direct  result  of 
metabolic  changes  brought  about  by  bacteria  in  the 
medium  or  tissues  in  which  they  may  be  developing — 
*.  e.j  they  are  products  of  nutrition  that  pass  readily  into 
solution,  as  is  conspicuously  seen  in  the  case  of  the 
bacillus  of  diphtheria  and  of  tetanus  when  under  both 
artificial  cultivation  and  in  the  animal  body.  Many 
bacteria  which  do  not  possess  the  power  of  generating 
or  secreting  such  poisons  may,  nevertheless,  have  inti- 
mately associated  with  their  protoplasmic  bodies  poison- 
ous substances  that  can  only  be  isolated  by  particular 
methods ;  thus  the  toxins  of  bacillus  tuberculosis  and  of 
spirillum  cholerce  Asiatics  are  much  more  conspicuously 
present  in  the  protoplasm  of  these  bacteria  than  in  the 
fluids  in  which  they  have  grown ;  and  Buchner  has 
isolated  from  several  species  of  bacteria  "bacterio- 
proteins"  having  the  common  properties  of  solubility 
in  alkalies,  resistance  to  the  boiling  temperature,  attrac- 
tion of  leucocytes  (positive  chemotaxis 2),  and  pyogenic 
powers. 

There  is  as  yet  little  agreement  of  opinion  as  to  the 
chemical  nature  of  toxins ;  but  it  is  probable  that  the 

1  The  term  "toxins"  is  commonly  applied  to  amorphous,  nitrogenous 
poisons  produced  by  bacteria  in  both  living  tissues  and  dead  substances ; 
while,  on  the  other  hand,  "ptomams"  relates  to  crystallizable,  nitrog- 
enous poisons  that  are  formed  in  dead  tissue,  and  "leucomains"  to 
poisonous  and   non-poisonous  alkaloidal   bodies  that  occur  in  living 
tissues  as  a  result  of  physiological  metabolism. 

2  See  Chemotaxis. 


INFECTION  AND  IMMUNITY.  495 

group  comprises  different  bodies  of  the  nature  of  globu- 
lins, nucleo-albumins,  peptones,  albumoses,  and  enzymes 
or  ferments. 

Toxic  ptomai'ns  are  probably  not  conspicuously  con- 
cerned in  producing  the  characteristic  symptoms  of 
infection,  as  they  are  absent  from  cultures  of  certain 
highly  pathogenic  bacteria. 

In  some  instances  the  production  of  poisonous  prin- 
ciples, even  under  artificial  conditions  of  cultivation,  is 
most  astonishing,  and  poisons  result  that  in  the  degree 
of  their  toxicity  exceed  anything  hitherto  known  to  us. 
For  instance,  the  potencies  of  the  poisons  that  have 
been  isolated  from  cultures  of  bacillus  diphtherm  and 
of  the  bacillus  of  tetanus  have  been  carefully  deter- 
mined by  experiments  upon  animals,  and  it  has  been 
found  that  0.4  milligramme  of  the  former  is  capable  of 
killing  eight  guinea-pigs,  each  weighing  400  grammes, 
or  two  rabbits,  each  weighing  3  kilogrammes  (Roux  and 
Yersin l) ;  and  that  0.0001  milligramme  of  the  latter 
will  produce  tetanus  in  a  mouse,  with  all  the  character- 
istic manifestations  of  the  disease  (Brieger  and  Cohn 2).3 

Briefly  stated,  the  invasion  of  the  body  by  infective 
micro-organisms  may  best  be  conceived  as  a  contest 
between  the  invading  organisms  on  the  one  side  and 
the  resisting  tissues  of  the  animal  body  on  the  other ; 
the  weapons  of  offence  of  the  former  being  the  poison- 
ous products  of  their  growth,  while  the  means  of 
defence  possessed  by  the  latter  are  vital  substances 

1  Annales  de  1'Institut  Pasteur,  1889,  tome  iii.  p.  287. 

2  Zeitschrift  fur  Hygiene  und  Infektionskrankheiten,  1893,  Bd.  xv. 
Heft  1. 

3  By  the  use  of  more  recently  devised  methods  we  are  enabled  to 
increase  still  further  the  toxicity  of  these  poisons;  especially  is  this 
the  case  with  regard  to  the  diphtheria  toxin. 


496  BACTERIOLOGY. 

which  act,  so  to  speak,  as  antidotes  to  these  poisons. 
If  the  tissue-elements  are  not  of  sufficient  vigor  to 
destroy  the  invading  bacteria  or  to  render  inert  the 
poisons  produced  by  them,  the  bacteria  are  victorious 
and  infection  results;  on  the  other  hand,  if  there  be 
failure  to  excite  a  condition  of  disease,  the  tissues  are 
victorious,  and  are  then  said  to  be  resistant  to  or  immune 
from  this  particular  form  of  infection. 

In  some  cases  the  animal  antidotes  act  directly  upon 
the  invading  organisms  themselves — i.  e.,  they  are 
germicidal ;  in  others  their  function  is  more  that  of 
antidotes,  or  neutralizers  in  the  chemical  sense,  for  the 
poisons  produced  by  these  organisms,  the  organisms 
themselves,  in  certain  instances,  experiencing  only 
slight  injury  from  a  limited  sojourn  in  the  living  tissues. 
To  the  substances  possessing  the  germicidal  functions 
that  are  normally  present  in  the  animal  body  Buchner 
gives  the  name  "  alexins,"  and  Hankin  "  defensive 
proteids ;"  while  to  those  ill-defined  elements  of  the 
body  that  act  more  directly  upon  the  soluble  bacterial 
poisons  the  name  "  antitoxins  "  is  now  generally  applied. 
Unlike  the  alexins  of  Buchner  and  the  defensive 
proteids  of  Hankin,  it  is  only  occasionally  that  anti- 
toxic activities  are  demonstrable  in  normal  living 
tissues  as  a  congenital  characteristic  ;!  in  the  majority 
of  cases  they  are  conspicuously  present  only  as  an 
acquired  peculiarity.  Our  acquaintance  with  these 
bodies  extends  little  further  than  their  physiological 
functions  and  some  of  the  means  that  induce  their 

1  See  Bolton :  Transactions  of  Association  of  American  Physicians, 
1896,  vol.  xi.  p.  62.  Pfeiffer :  Deutsche  med.  Wochenschrift,  1896,  No.  8. 
Fischl  and  v.  Wunschheim :  Centralblatt  fur  Bakteriologie,  Parasiten- 
kunde,  und  Infektionskrankheiten,  1896,  Abt.  i.  Bd.  xix.  S.  652. 
Wassermann  :  Berliner  klin.  Wochenschrift,  1898,  No.  1. 


INFECTION  AND  IMMUNITY.  497 

generation.  We  have  no  satisfactory  knowledge  of 
their  intimate  nature  or  of  the  primary  sources  of 
their  production.  They  are  believed  by  some  (Buch- 
ner1  and  Metchnikoff2)  to  represent  bacterial  toxins 
that  have  been  converted  through  the  vital  activity  of 
the  integral  cells  of  the  body ;  and  Roux  s  and  Buch- 
ner4  hold  that  they  exhibit  their  protective  functions 
less  by  direct,  destructive  combination  with  the  toxins 
than  by  a  specific  stimulation  of  the  tissue-cells  that 
enables  the  latter  to  resist  the  harmful  influences  of 
the  poisonous  bacterial  products.  On  the  other  hand, 
Behring,5  Ehrlich,6  and  their  associates  contend  that 
they  are  vital  tissue-elements  ("  toxophores,"  Ehrlich), 
having  the  property  of  combining  directly  with  the 
toxins  to  form  "physiologically  neutral"  toxin-anti- 
toxin compounds  that  are  in  a  manner  analogous  to 
the  double  salts  of  certain  chemical  reactions. 

It  is  a  common  observation  that  many  human  beings 
and  animals  are  less  susceptible  to  the  different  forms 
of  infection  than  are  others ;  and  that  particular  species 
of  animals  not  only  do  not  suffer  naturally  from 
certain  specific  diseases,  but  resist  all  efforts  to  pro- 
duce the  diseases  in  them  by  artificial  methods ; 
in  other  words,  they  are  naturally  immune  from 
them.  The  term  "natural  immunity,"  as  now  em- 
ployed, implies  a  congenital  condition  of  the  indi- 
vidual or  species,  a  condition  peculiar  to  his  idioplasm, 

1  Bachner :  Munchener  med.  Wochenschrift,  1893,  Nos.  24  and  25. 

2  Metchnikoff:  Weil's  Handbuch  der  Hygiene,  Bd.  ix.  Lieferung  1. 

3  Koux :  Annales  de  1'Institut  Pasteur,  1894,  p.  722. 

4  Buchner:  Berliner  klin.  Wochenschrift,  1894,  No.  4. 

5  Behring :  Infektion  und  Desinfektion,  Leipzig,  1894,  S.  248. 

•  Ehrlich :  Klinisches  Jahrbuch,  1897,  Bd.  vi.  Heft  2,  S.  311.     Fort- 
schritte  der  Medicin,  1897,  Bd.  xv.  No.  2. 
32 


498  BACTERIOLOGY. 

which  has  been  transmitted  to  him  as  a  tissue-charac- 
teristic through  generations  of  progenitors. 

Again,  it  is  often  observed  that  an  individual  or  an 
animal  after  having  recovered  from  certain  forms  of 
infection  has  thereby  acquired  protection  from  subse- 
quent attacks  of  like  character;  in  other  words,  they 
are  said  to  have  acquired  immunity  from  this  disease. 
"Acquired  immunity"  implies,  therefore,  a  condition 
of  the  tissues  of  an  individual,  not  of  necessity  peculiar 
to  other  members  of  the  race  or  species,  that  has 
originated  during  his  life  from  the  stimulation  of  his 
integral  cells  by  one  or  another  of  the  specific,  infective 
irritants  that  may  have  been  purposely  introduced,  or 
accidentally  gained  access  to  his  body. 

The  problem  involving  the  explanation  of  the  fore- 
going interesting  ideas  and  observations  has  afforded 
material  for  reflection  and  hypothesis  for  a  long  time. 
It  is  only  through  investigations  conducted  during  re- 
cent years  that  it  has  met  with  anything  approaching 
satisfactory  solution,  and  even  now  there  remain  a 
number  of  important  points  that  are  veiled  in  obscurity. 

Conspicuous  among  the  observers  who  have  endeav- 
ored to  explain  the  modus  operandi  of  immunity  may 
be  mentioned  Chauveau,  Pasteur,  Metchnikoff,  Buch- 
ner,  Flugge  and  his  pupils  (Smirnow,  Sirotinin,  Bitter, 
Nuttall),  Fodor,  Hankin,  Pfeiffer,  Ehrlich,  Behring, 
Roux,  and  a  host  of  others  whose  names  are  more  or 
less  prominent  in  current  literature.  In  the  following 
pages  we  will  present  briefly  the  result  of  investigations 
by  these  various  authors. 

In  1880  Chauveau  l  suggested  an  explanation  for  the 
phenomenon  of  immunity  that  has  since  been  known  as 

1  Comptes  rendus,  etc.,  July,  1880,  No.  91. 


INFECTION  AND  IMMUNITY.  499 

the  " retention  hypothesis"  It  is,  in  short,  as  follows : 
that  the  immunity  commonly  seen  to  exist  in  animals 
that  have  passed  through  an  attack  of  infection  from 
a  subsequent  outbreak  of  the  same  malady,  and  likewise 
the  immunity  that  has  been  produced  artificially  by 
vaccination,  exist  by  virtue  of  some  bacterial  product 
that  has  been  retained  or  deposited  in  the  tissues  of  those 
animals,  and  that  this  product  by  its  presence  prevents 
the  development  of  the  same  organisms  if  they  should 
subsequently  gain  access  to  the  body. 

Bearing  upon  this  view  the  experiments  of  Sirotinin,1 
made  with  cultures  of  various  pathogenic  bacteria, 
demonstrated  that,  in  so  far  as  culture-experiments  were 
concerned,  the  only  substance  produced  by  growing 
bacteria  that  could  be  in  any  way  inimical  to  their  further 
development  were  substances  that  gave  rise  to  altera- 
tions in  the  reaction  of  the  medium  in  which  they  were 
developing — i.  e.,  acids  or  alkalies  produced  by  the  bac- 
teria themselves.  So  long  as  the  organisms  were  not 
actually  dead  from  exposure  to  these  substances  correc- 
tion of  the  abnormal  reaction  was  followed  by  further 
development  of  the  organisms.  Sirotinin  also  states 
that  materials  containing  the  products  of  growth  of 
bacteria,  so  long  as  they  are  maintained  at  a  neutral  or 
onlv  slightly  alkaline  reaction,  serve  very  well  as  media 
upon  which  to  cultivate  again  the  same  organism  that 
produced  them,  providing  the  nutritive  elements  have 
not  been  entirely  exhausted.  He  remarks  that,  if  in  such 
a  concentrated  form  as  we  find  the  life-products  of  bac- 
teria in  the  medium  in  which  they  are  growing,  no  inhib- 
itory compounds  other  than  acids  and  alkalies  are  to 
be  detected,  it  is  hardly  probable  that  they  are  produced 

1  Zeitscbrift  fur  Hygiene,  1888,  Bd.  iv. 


500  BACTERIOLOGY. 

in  the  tissues  of  the  living  animal,  and  retained  there, 
to  a  degree  sufficient  to  prevent  the  growth  of  bacteria 
that  may  subsequently  gain  entrance  to  these  tissues, 
after  the  disappearance  of  the  organisms  concerned  in 
the  primary  invasion.  On  the  other  hand,  Salmon  and 
Smith,1  Roux  and  Chamberland,2  and  others  had 
demonstrated  that  a  sort  of  immunity  against  certain 
forms  of  infection  may  be  afforded  to  susceptible  ani- 
mals by  the  injection  into  their  tissues  of  the  products 
of  growth  of  particular  organisms  which  would,  if  them- 
selves introduced  into  the  animal  body,  produce  fatal 
results.  Though  this  observation  of  Salmon  and  Smith 
attracted  comparatively  little  attention  at  the  time  it 
was  made,  it  serves,  nevertheless,  as  we  shall  see  sub- 
sequently, as  the  starting-point  for  a  line  of  investigation 
that  has  furnished  practically  all  the  information  of  im- 
portance that  we  possess  on  this  complicated  subject. 

As  opposed  to  the  view  of  Chauveau,  Pasteur3  and 
certain  of  his  pupils  believed  that  the  immunity  fre- 
quently afforded  to  the  tissues  by  an  attack  of  infection 
or  following  upon  vaccination  against  infection,  was 
due  rather  to  an  abstraction  from  the  tissues,  by  the 
organisms  that  were  concerned  in  the  primary  attack, 
of  a  something  that  is  necessary  to  the  growth  of  the 
infecting  organism  should  it  gain  entrance  to  the  body 
at  any  subsequent  time.  This  view  is  known  as  the 
"  exhaustion  hypothesis." 

As  to  the  exhaustion  hypothesis  of  Pasteur,  there  is, 
as  yet,  no  evidence  whatever  for  its  support ;  and,  in 

1  Proceedings  of  the  Biological  Society,  Washington,  D.  C.,   1886, 
vol.  iii. 

2  Annales  de  1'Institut  Pasteur.  1888-'89,  tomes  i.,  ii. 

3  Bulletin  de  PAcademie  de  Medeciue,  1880. 


INFECTION  AND  IMMUNITY.  501 

fact,  in  the  light  shed  by  more  recent  investigations  this 
is  probably  the  least  tenable  of  any  of  the  several  hy- 
potheses advanced  in  explanation  of  this  most  com- 
plicated phenomenon.  The  work  of  Bitter,1  which  was 
undertaken  with  the  view  of  determining  if,  in  the  proc- 
ess of  acquiring  immunity,  there  occurred  this  ex- 
haustion from  the  tissues  of  material  necessary  to  the 
growth  of  bacteria  that  might  gain  entrance  to  them  at 
some  later  date,  gave  only  negative  results.  The  flesh 
of  animals  in  which  immunity  had  been  produced  con- 
tained all  the  elements  necessary  for  the  growth  and 
nutrition  of  the  bacteria  against  which  the  animals  had 
been  protected,  just  as  did  the  flesh  of  non- vaccinated 
animals. 

1884  Metehnikoif2  published  the  first  of  a  series 
of  observations  upon  the  behavior  of  certain  of  the 
mesodermal  cells  of  lower  animals  toward  insoluble 
particles  that  may  be  present  in  the  tissues  of  these 
animals.  The  outcome  of  these  investigations  was  the 
establishment  of  his  well-known  doctrine  of  phago- 
cytosis, the  principle  of  which  is  that  the  wandering 
cells  of  the  animal  organism,  the  leucocytes,  possess 
the  property  of  taking  up,  rendering  inert,  and  di- 
gesting micro-organisms  which  they  may  encounter 
in  the  tissues.  Metchnikoff  believed  that  in  this 
way  immunity  from  infection  may  in  many,  if  not 
all,  cases  be  explained.  He  believed  that  suscepti- 
bility to  or  immunity  from  infection  was  essentially  a 
matter  between  the  invading  bacteria  on  the  one  hand, 
and  the  leucocytes  of  the  tissues  on  the  other.  The  suc- 

1  Zeitschrift  fur  Hygiene,  1888,  Bd.  iv. 

2  Arbeiten  aus  dem   Zoologischen    Institut  der  Universitat  Wien, 
1884,  Bd.  v.    Fortschritte  der  Medicin,  1884,  Bd.  ii. 


502  BACTERIOLOGY. 

cess  or  failure  of  the  leucocytes  in  protecting  the  animal 
against  infection  depends,  according  to  this  doctrine, 
entirely  upon  the  efficiency  of  the  provisions  possessed 
by  them  for  destroying  bacteria.  When  these  means 
are  of  sufficient  vigor  to  bring  about  the  death  of  the 
bacteria  the  tissues  are  victorious ;  but  when  the  poisons 
generated  by  the  bacteria  are  potent  to  arrest  the  pha- 
gocytic  action  of  the  leucocytes -then  the  tissues  succumb 
and  infection  results. 

Has  this  doctrine  of  phagocytosis,  as  advanced  by 
Metchnikoff,  stood  the  test  of  experimental  criticism? 
Evidence  that  has  accrued  since  the  time  of  its  sugges- 
tion has  rendered  questionable  the  advisability  of  its 
unconditional  adoption  in  the  sense  that  Metchnikoif 
propounded  it.  The  later  studies  of  a  number  of  in- 
vestigators indicate  that  while  the  leucocytes  play  a 
most  important  part  in  the  phenomenon  of  immunity, 
it  is  hardly  likely  that  this  always  occurs  through  their 
taking  up  within  themselves  and  digesting  invading 
bacteria,  as  Metchnikoif  believes ;  but  rather  that  their 
part  in  the  process  is  to  secrete  protective  chemical  sub- 
stances that  are  thrown  into  the  circulating  blood,  and 
which,  in  part  at  least,  comprise  the  defensive  bodies  to 
which  Buchner  has  given  the  name  "  alexins."  l 

The  first  severe  blow  that  MetchnikofPs  theory  of 
phagocytosis  received  was  given  by  Nuttall,2  in  his 
work  upon  the  bactericidal  property  of  the  animal  econ- 
omy. In  these  experiments  Nuttall  showed  positively 
that  the  destruction  of  virulent  bacteria  in  the  blood 
of  animals  was  not  necessarily  dependent  upon  the 
actual  presence  of  living  leucocytes,  but  that  the  serum 

1  See  Hahn  :  Archiv  fur  Hygiene,  1895,  Bd.  xxv,  S.  105, 

2  Zeitschrift  fur  Hygiene,  1888,  Bd.  iv, 


INFECTION  AND  IMMUNITY.  503 

of  the  blood  when  quite  free  from  cellular  elements 
possessed  this  power  to  a  degree  equal  to  that  of  the 
blood  wheu  all  the  constituent  parts  were  present.  In 
the  blood,  as  such,  phagocytosis  could  be  seen  ;  but,  as  a 
rule,  the  bacteria  presented  evidence  of  having  under- 
gone degenerative  changes  before  they  had  been  taken 
up  by  the  wandering  cells — i.  e.,  the  bacteria  had  evi- 
dently been  injured  or  killed  by  the*  fluids  before  they 
were  attacked  by  the  phagocytes. 

Contrary  to  the  beliefs  in  existence  at  the  time, 
Traube  and  Gscheidlen,1  as  far  back  as  1874,  demon- 
strated that  considerable  quantities  of  septic  material 
could  be  injected  into  the  circulation  of  warm-blooded 
animals  without  apparently  any  effect  upon  the  animal. 
Particularly  was  this  the  case  with  dogs.  If  they  in- 
jected into  the  circulation  of  a  dog  as  much  as  1.5  c.c. 
of  decomposing  fluid,  blood  drawn  from  the  animal 
after  from  twenty-four  to  forty-eight  hours  showed  no 
special  tendency  to  decompose,  though  it  was  kept 
under  observation  for  a  long  time.  As  a  result  of  these 
experiments,  the  question  that  naturally  presented  itself 
was :  Does  the  animal  organism  possess  the  power  of 
rendering  septic  organisms  inert,  and  if  so,  to  what 
extent  ?  They  believed  this  power  of  rendering  living 
organisms  inert  to  be  possessed  by  the  circulating 
blood  to  only  a  limited  degree,  for  after  the  injection 
of  much  larger  amounts  of  the  putrid  fluid  into  the 
blood  of  the  animal  death  usually  ensued  in  from 
twenty-four  to  forty-eight  hours.  The  blood  drawn 
from  the  animal  just  before  death  contained  the  living 
bacteria  of  putrefaction  and  underwent  decomposition. 
They  attributed  the  germicidal  phenomenon  to  the 
1  Jahresb.  der  Schlesischen  Ges.  fiir  Cultur.,  1874 ;  Jahr.  iii.  p.  179. 


504  BACTERIOLOGY. 

action  of  the  "  ozonized  oxygen  of  the  corpuscles  of  the 
blood." 

Similar  observations  were  made  in  1885  by  Fodor,1 
who  remarks  upon  the  rapidity  with  which  living  bac- 
teria disappear  from  the  circulating  blood  of  animals ; 
and  Wyssokowitsch,2  who  endeavored  to  explain  this 
disappearance  experimentally,  went  wide  of  the  mark 
by  concluding  that'  they  were  filtered  from  the  blood 
and  digested  by  the  parenchymatous  tissues. 

In  1882  Rauschenbach 3  demonstrated  that  when  in 
the  process  of  coagulation  fibrin  was  formed,  it  was  not 
as  a  specific  product  of  the  action  of  the  colorless  ele- 
ments of  the  blood  alone,  but  also  as  a  result  of  the 
combined  action  between  all  animal  protoplasms  and 
healthy  blood-plasma,  and  that  in  the  process  there  was 
always  a  disintegration  of  the  leucocytes  present.  In 
1884  Groth 4  demonstrated  further  that  such  a  disinte- 
gration of  leucocytes  occurred  in  normal  circulating 
blood,  though  here  it  was  not  accompanied  by  coagula- 
tion. The  results  of  these  observations  suggested  the 
question  :  Does  such  a  disintegration  occur  when  vege- 
table protoplasm  is  introduced  into  the  blood  ? 

For  the  purpose  of  answering  this  question,  Grohmann,5 
a  pupil  of  Alexander  Schmidt,  undertook  to  study  the  ac- 
tion of  the  circulating  blood  upon  the  vegetable  proto- 
plasm of  bacteria.  He  noticed  that  clotting  of  the  blood 

1  Fodor :  Archiv  fur  Hygiene,  Bd.  iv.  S.  129. 

2  Wyssokowitsch :  Zeitschrift  fur  Hygiene,  1886,  S.  3. 

3  Ueber  die  Wechselwirkung  zwischen  Protoplasma  und  Blutplasma. 
Dissertation,  Dorpat,  1882. 

4  Ueber  die  Schicksale  der  farblosen  Elemente  in  kreisendem  Blut. 
Dissertation,  Dorpat,  1884. 

6  Ueber  die  Einwirkung   des   zellenfreien    Blutplasma  auf   einige 
pflanzliche  Mikro-organismen,     Dissertation,  Dorpat,  1884. 


INFECTION  AND  IMMUNITY.  505 

of  the  horse  was  very  much  accelerated  by  the  addition  to 
it  of  certain  bacteria ;  that  at  the  same  time  the  develop- 
ment of  the  bacteria  was  checked,  and  in  the  case  of  the 
pathogenic  varieties  their  virulence  was  diminished. 
This  was  particularly  the  case  when  the  anthrax  bacil- 
lus was  employed. 

Grohrnann  seems  to  have  appreciated  the  significance 
of  this  observation,  though  he  made  no  attempt  to  study 
the  subject  more  closely.  He  remarks  that  the  system 
probably  possesses,  in  the  plasma  of  the  blood,  a  body  hav- 
ing disinfectant  properties.1  His  observations  on  this 
particular  point  were,  however,  only  incidental,  and  it 
was  not  until  the  researches  of  Nuttall,  directed  especi- 
ally toward  the  question  of  germicidal  properties  of 
normal  animal  fluids,  that  a  complete  and  acceptable 
demonstration  of  this  important  function  was  forthcom- 
ing, and  that  a  general  interest,  commensurate  with  its 
importance,  was  created  in  the  subject. 

Nuttall's  results  received  confirmation  from  all  sides. 
Fodor,2  Buchner,3  Lubarsch,4  Nissen,5  Stern,6  Prudden,7 
Charrin  and  Roger,8  and  many  others  continued  in  the 
same  line,  and  all  made  practically  the  same  observation. 

After  the  demonstration  by  Kuttall  that  the  serum 
of  the  blood  was  directly  detrimental  to  the  vitality  of 
certain  pathogenic  bacteria,  a  number  of  investigators 
undertook  to  determine  the  conditions  most  favorable  to 

1  Loc.  cit,  pp.  6  and  33. 

2  Central,  fur  Bakteriologie  und  Parasitenkunde,  1890,  Bd.  vii.  No.  24. 

*  Archiv  fur  Hygiene,  1890,  vol.  x.  parts  1  and  2. 

*  Centralblatt  fur  Bakteriologie  und  Parasitenkunde,  1889,  Bd.  vi. 
No.  18. 

6  Zeitschrift  fur  Hygiene,  1889,  Bd.  vi.  part  3. 

6  Zeitschrift  fur  klin.  Medicin,  1890,  Bd.  viii.  parts  1  and  2. 

7  New  York  Medical  Record,  1890,  vol.  xxxvii.  pp.  85  and  86. 

8  Societe  de  Biologic  de  Paris. 


506  BACTERIOLOGY. 

the  exhibition  of  this  phenomenon,  and  further  to  de- 
cide upon  the  constituent  of  the  serum  to  which  this 
property  is  due,  or  if  it  is  a  function  of  the  serum  only 
as  a  whole. 

In  the  course  of  Buchner's  experiments  it  was  de- 
monstrated that  the  serum  was  robbed  of  this  property 
by  exposure  to  a  temperature  of  55°  C.  for  half  an 
hour ;  that  its  efficacy  as  a  germicide  was  not  dimin- 
ished by  alternate  freezing  and  thawing ;  that  by  dialy- 
sis or  extreme  dilution  with  distilled  water  its  germicidal 
activity  was  diminished  or  completely  checked ;  but 
that  an  equal  dilution  could  be  made,  if  sodium  chlo- 
ride solution  (0.6-0.7  per  cent.)  was  substituted  for  the 
distilled  water,  without  a  reduction  of  its  bactericidal 
activity.  From  this  he  concluded  that  the  active  ele- 
ment in  this  phenomenon  is  a  living  albumin,  an  essen- 
tial constituent  of  which  is  sodium  chloride,  and  which, 
when  robbed  of  this  salt  either  by  dialysis  or  dilution, 
becomes  inert  in  its  behavior  toward  bacteria.  For  this 
or  these  germicidal  constituents  of  the  blood  he  sug- 
gested the  name  "  alexins." 

Buchner  found,  moreover,  that  the  activity  of  the 
serum  alone  against  bacteria  was  greater  than  when  the 
cellular  elements  of  the  blood  were  present.  This  he 
explains  by  the  assumption  that  in  the  serum  alone  the 
germicidal  element  predominates,  whereas  in  the  blood, 
as  such,  outside  of  the  body,  it  is  still  present,  but  its 
influence  is  counteracted  by  the  nutrition  offered  to  the 
bacteria  by  the  disintegrated  cellular  elements  ;  so  that 
here  the  nutritive  feature  is  most  conspicuous  and  the 
destructive  activity  toward  bacteria  is  less  effectual. 

A  closer  study  of  the  nature  of  this  germicidal  ele- 
ment in  the  body  of  animals  was  made  by  Hankin  and 


INFECTION  AND  IMMUNITY.  507 

Martin.1  The  former  isolated  from  the  spleen  and 
lymphatic  glands  a  body — a  globulin — which  in  solu- 
tion possesses  germicidal  properties. 

Similar  germicidal,  ferment-like  globulins  have  been 
isolated  from  the  blood  by  Ogata,2  and  in  their  studies 
upon  tetanus  Tizzoni  and  Cattani 3  found  a  body  that 
was  antagonistic  to  the  poison  produced  by  the  organism 
of  this  disease. 

In  1890  Fodor4  showed  that  the  fatal  results  of  an- 
thrax infection  could  be  materially  retarded  by  alkalini- 
zation  of  the  circulating  blood ;  and  he  further  states 
that  it  is  occasionally  possible  by  the  administration  of 
alkalies  per  os  to  rescue  animals  already  infected. 

According  to  the  observations  of  Yaughau,5  the  most 
important  germicidal  or  protective  agents  possessed  by 
the  body  are  the  nucleins ;  and  Kossel  has  shown  that 
the  cholera  vibrio,  streptococcus,  staphylococcus,  and 
typhoid  bacillus  are  destroyed  by  0.5  per  cent,  solution 
of  nucleinic  acid. 

Hankin  believes  the  globulins  or  "defensive  pro- 
teids"  that  he  has  discovered  and  the  albuminoid 
bodies  studied  by  Buchner  to  be  identical.  The  most 
interesting  and,  in  the  light  of  work  that  has  appeared 
since,  the  most  important,  of  Hankin's  observations 
were  not  those  upon  the  power  of  these  globulins  to 
destroy  the  vitality  of  living  organisms,  but  rather 
those  upon  the  relation  between  them  and  the  poison- 
ous metabolic  products  of  growth  of  the  organisms. 

1  British  Medical  Journal,  May  31, 1890. 

s  Centralblatt  fur  Bakteriologie  und  Parasitenkunde,  1891,  vol.  ix 
8.599. 

3  Ibid.,  p.  685. 

4  Fodor :  Centralblatt  fur  Bakteriologie,  1890,  Bd.  vii.  S.  753-766. 

5  Vaughan  :  Medical  News,  May  20,  1893. 


508  BACTERIOLOGY. 

For  example,  if  the  poisonous  products  of  virulent 
anthrax  bacilli  be  isolated  and  mixed  with  the  glob- 
ulin extracted  from  normal  tissues,  the  experiments  of 
Hankin  showed  a  directly  destructive  action  on  the  part 
of  the  bacterial  products.  He  found  that  the  amount  of 
poisonous  albumose  produced  by  the  attenuated  anthrax 
bacilli  employed  as  vaccines  was  much  less  than  that 
produced  by  organisms  possessing  full  virulence ;  and 
he  suggests  that  perhaps  the  protective  influence  of 
vaccinations  that  are  practised  by  introducing  into  the 
animal  the  organisms  that  have  been  attentuated  in  vir- 
ulence is  due  to  a  gradual  tolerance  acquired  by  the 
cells  of  the  tissues  to  the  action  of  the  poison  when 
produced  in  these  small  quantities ;  in  the  same  way 
that  a  tolerance  was  acquired  by  the  tissues  for  the 
venom  of  the  rattlesnake  in  the  experiments  of  Sewall  * 
(and  more  recently  in  the  work  of  Eraser,2  Calmette, 
Weir  Mitchell,  and  others),  and  similar  to  that  follow- 
ing the  injection  into  the  tissues  of  small  quantities  of 
hemialbumose,  which  in  large  amounts  rapidly  proves 
fatal. 

Of  utmost  importance  to  these  investigations  upon 
the  blood  and  fluids  of  the  body  are  the  experiments 

1  Journal  of  Physiology,  1887,  vol.  vii.  p.  203. 

2  One  of  the  important  results  of  Eraser's  studies  is  the  demonstra- 
tion that  the  bile  of  normal  serpents  and  of  certain  warm-blooded 
animals,    notably  bovines,   rabbits,  and   guinea-pigs,  is  antitoxic  for 
serpent  venom.    The  antitoxic  value  of  the  bile  in  this  connection 
seems  to  be  fairly  comparable  to  that  of  other  antitoxins  obtained 
from  the  blood  of  artificially  immunized  animals.     Its  action  is  not 
only  prophylactic,   but  also  curative,   Fraser    having   succeeded   in 
rescuing  animals  by  the  injection  of  bile-extract  thirty  minutes  after 
they  had  received  what  would  otherwise  have  been  a  fatal  dose  of 
cobra  poison.     (See  Centralblatt  fiir  Bakteriologie,  1897,  Bd.  xxii.  S. 
420.) 


INFECTION  AND  IMMUNITY.  509 

of  Behring  and  Kitasato  l  upon  the  production  of  im- 
munity from  tetanus.  In  their  studies  upon  the  blood 
of  animals  subjected  to  these  experiments  it  was  found 
that  it  was  not  only  possible  to  render  animals  immune 
from  this  disease,  but  that  the  serum  of  the  blood  of 
these  immunized  animals  afforded  immunity  when  in- 
jected into  the  peritoneal  cavity  of  other  animals  that 
had  not  been  so  protected ;  and  moreover,  that  in  some 
cases  this  serum  possesses  curative  powers  over  the 
disease  after  it  has  been  in  progress  for  a  time.  They 
found,  further,  that  the  serum  of  animals  that  had  been 
rendered  immune  from  tetanus,  when  brought  in  con- 
tact with  the  poison  of  tetanus,  completely  destroyed  its 
poisonous  properties,  and  that  the  serum  from  animals 
or  from  human  beings  that  do  not  possess  immunity 
from  this  disease  has  no  such  power. 

The  demonstration  by  Behring  and  Kitasato  of  the 
fact  that  the  serum  of  an  immunized  animal  can  not 
only  confer  immunity  upon  another  susceptible  animal, 
but  in  the  case  of  tetanus  (and  diphtheria,  as  subse- 
quently demonstrated  by  Behring  and  his  associates), 
cure  the  disease  after  it  is  already  in  progress,  is  one  of 
the  most  important  steps  that  has  been  made  in  this 
entire  field  of  study.  The  application  of  the  principle 
involved  in  this  observation  to  the  cure  of  diphtheria 
in  man  has  resulted  in  a  triumph  which  marks  an  epoch 
in  modern  scientific  medicine.  The  same  principle  has 
been  employed  for  obtaining  curative  agents  against 
other  forms  of  infection  and  intoxication,  notably,  of 
Asiatic  cholera,  typhoid  fever,  lobar  pneumonia,  strep- 
tococcus and  staphylococcus  infection,  rabies,  tuberculosis, 

1  Behring  und  Kitasato :    Deutsche  med.  Wochenschrift,  1890,  Bd. 
xvi.  S.  1113. 


510  BACTERIOLOGY. 

bubonic  plague,  syphilis,  vaccinia,  and  serpent-venom ; 
but  unfortunately,  as  yet,  with  only  indifferent  success ; 
certainly  in  no  case  to  the  same  favorable  degree  as  has 
been  seen  in  the  treatment  of  diphtheria  with  antitoxic 
serum. 

Another  hypothesis  in  explanation  of  the  immunity 
acquired  by  the  tissues  of  the  animal  organism  is  that 
advanced  by  Buchner,1  who  suggests  that  in  the  primary 
infection,  from  which  the  animal  has  recovered,  there 
has  been  produced  a  reactive  change  in  the  integral  cells 
of  the  body  that  enables  them  to  protect  themselves 
against  subsequent  inroads  of  the  same  organism. 
Though  somewhat  more  vague  at  first  glance  than  the 
other  theories  in  regard  to  this  phenomenon,  it  has, 
nevertheless,  much  to  recommend  it,  and  in  the  light 
of  subsequent  research  is  regarded  by  many  as  prob- 
ably the  correct  explanation  of  the  establishment  of  im- 
munity in  a  number  of,  if  not  in  all,  cases.  Experiments 
that  bear  directly  upon  this  idea  have  demonstrated 
that,  if  animals  be  subjected  to  injections  of  the  poison- 
ous products  of  growth  of  certain  virulent  bacteria, 
they  respond  to  this  treatment  by  more  or  less  pro- 
nounced constitutional  reactions,  and  that  during  this 
period,  and  for  a  short  time  following,  they  are  protected 
from  the  invasion  of  the  virulent  bacteria  themselves. 
This  observation  has,  moreover,  not  been  confined  to 
those  cases  in  which  injections  of  the  products  of  growth 
were  followed  by  inoculations  with  the  bacteria  by 
which  they  were  produced,  but,  what  is  still  more  in- 
teresting and  confirmatory  of  Buchner's  view,  it  is 
claimed  that  a  sort  of  protection  from  certain  specific 

1  Buchner :  Eine  neue  Theorie  uber  Erzielung  vou  Immuuitat  gegen 
Infektionskraukheiten.  Muenchen,  1883. 


INFECTION  AND  IMMUNITY.  511 

infections  can  also  be  afforded  to  animals  by  the  injec- 
tion into  them  of  cultures  of  entirely  different  species  of  -, 
bacteria,  or  their  products,  and  that  in  some  cases  these 
are  not  of  necessity  of  the  disease-producing  variety.  ^ 
For  instance,  Emmerich  and   Mattei 1  claim  to  have 
rendered  rabbits  insusceptible  to  anthrax  through  injec- 
tions into  them  of  cultures  of  the  streptococcus  of  ery- 
sipelas. 

This,  they  claim,  is  not  due  to  any  antagonism  be- 
tween the  two  organisms  themselves,  for  in  culture 
experiments  they  grew  well  together,  without  any 
alteration  in  their  pathogenic  properties;  but  rather 
to  the  induction  of  a  tissue  activity  by  which  resistance 
to  the  inroads  of  the  virulent  bacilli  was  established. 
Emmerich  and  Mattei  interpret  this  "reactive  tissue- 
change"  as  a  power  acquired  by  the  integral  cells  of  the 
body,  through  the  influence  of  a  stimulus,  of  generating 
a  product  that  is  detrimental  to  the  pathogenic  activity 
of  the  anthrax  bacilli. 

Pawlowsky,2  who  obtained  similar  results  from  the 
introduction  into  animals  of  cultures  of  bacillus  pro- 
digiosus,  of  staphyloeoccus  pyogenes  aureus,  and  of  mi- 
vrococcus  lanceolatus,  believes  them  to  be  due  to  the 
induction  of  increased  energy  on  the  part  of  the  wan- 
dering cells,  preparing  them  thus  for  the  difficult  task 
of  destroying  the  more  virulent  organisms  with  which 
the  animal  is  subsequently  to  be  inoculated. 

Protection  afforded  in  this  way  apparently  contra- 
indicates  a  specific  relation  between  the  morbific  ele- 
ments of  particular  infections  and  the  protective  sub- 
stances that  are  present  in  the  body  of  the  animal  that 

1  Emmerich  tmd  Mattei :  Fortschritte  der  Medicin,  1887,  S.  653. 

2  Pawlowsky  :  Virchow's  Archiv,  vol.  cviii.  p.  494. 


512  BACTERIOLOGY. 

has  been  rendered  insusceptible  to  them.  It  is  proba- 
ble, however,  that  this  is  only  apparent,  and  that  the 
observations  of  Emmerich  and  Mattei  and  of  Paw- 
lowsky  can  be  interpreted  in  another  way  :  in  the  blood 
of  animals  there  is  present  what  may  be  termed  a  nor- 
mal protective  (Buchner's  alexins)  having  no  specific  re- 
lations to  any  particular  variety  of  infection,  but  serving 
to  protect  the  animal  more  or  less  completely  against  all 
bacterial  invasion.  By  the  methods  employed  in  the 
preceding  experiments  it  seems  likely,  in  the  light  of 
more  recent  work,  that  this  normal  antidote  was  simply 
temporarily  accentuated  through  the  tissue-stimulation 
resultant  upon  the  treatment  that  the  animals  had  re- 
ceived, for  it  is  not  possible  to  bring  about  in  this  way 
as  high  or  as  permanent  a  degree  of  immunity  in  an 
animal  from  a  particular  disease  as  that  which  can  be 
obtained  by  the  use  of  the  specific  micro-organism  caus- 
ing the  disease,  or  the  products  of  its  growth,  especially 
the  latter. 

A  striking  illustration  of  this  protective  reaction  on 
the  part  of  the  animal  tissues  is  brought  out  in  the 
course  of  R.  Pfeiifer's T  experiments  on  Asiatic  cholera. 
He  found  that  it  was  possible  to  confer  immunity  upon 
animals  from  this  disease  ;  that  the  blood-serum  of  such  * 
animals  protected  susceptible  animals  into  which  it  was 
injected  against  what  would  otherwise  be  a  fatal  dose  of 
the  cholera  spirillum;  that  the  peritoneal  fluids  of  the 
artificially  immunized  animal  had  an  almost  instanta- 
neous disintegrating  (bacteriolytic),  bactericidal  action 
upon  living  cholera  spirilla  that  were  injected  into  the 
peritoneal  cavity ;  that  the  serum  from  the  immune 

1  Pfeiffer:  Zeitschrift  fiir  Hygiene  und  Infektionskrankheiten,  Bd. 
xviii.  S.  1 ;  Bd.  xx.  S.  198 


INFECTION  AND  IMMUNITY.  513 

animal  had  no  such  effect  upon  cholera  spirilla  in 
the  test-tube ;  but  when  virulent  cholera  spirilla  were 
injected  into  the  peritoneum  of  an  animal  that  was  not 
immune,  and  this  was  at  once  followed  by  an  intra- 
pcritoneal  injection  of  serum  from  an  immune  animal, 
almost  instantly  the  peculiar  disintegration  of  the  bac- 
teria, as  observed  in  the  peritoneum  of  the  immune  ani- 
mal, could  be  detected.  This  latter  observation  is  of 
the  utmost  importance  in  its  bearing  on  Buchner's 
hypothesis,  for  we  see  here  a  serum  from  an  immune 
animal  that  is  capable  of  conferring  immunity  ;  capable, 
on  injection  into  a  susceptible  animal,  of  endowing  its 
fluids  with  the  peculiar  disintegrating,  germicidal 
function  noted  in  the  peritoneum  of  the  immune  ani- 
mal from  which  the  serum  originated  ;  incapable  of 
bacteriolytic  activity  outside  the  body,  but  the  influ- 
ence of  which  in^tt^^erUoneum  of  a  susceptible  ani- 
mal is  to  call  forth  at  once  this  interesting  phenomenon. 
Manifestly  the  germicidal  substance  in  this  case  is 
neither  contained  in  the  protective  serum  nor  in  the 
peritoneum  of  the  susceptible  animal  before  receiving 
the  serum,  but  is  generated  by  the  tissues  as  a  result  of 
the  specific  irritation  of  a  something  contained  in  this 
serum  ;  in  other  words,  in  consequence  of  a  reaction  on 
the  part  of  the  peritoneal  tissues,  or  possibly  those  of 
the  entire  animal. 

In  more  recent  investigations  Pfeiffer,  in  association 
with  Marx,  has  found  that  the  antitoxic  substances  in 
cholera-immune  animals  are  much  more  abundant  in 
the  blood-building  organs — spleen,  lymphatic  glands, 
and  bone-marrow — than  in  either  the  blood  or  other 
tissues.1 

1  Pfeiffer  and  Marx  :  Deutsche  med.  Wochenschrift,  1898,  No.  3. 
33 


514  BACTERIOLOGY. 

The  experiments  of  G.  and  F.  Klemperer l  upon  acute 
fibrinous  pneumonia,  though  too  limited  in  extent  to 
be  accepted  as  conclusive,  presented  nevertheless  a 
number  of  most  significant  suggestions,  not  only  in 
connection  with  several  obscure  features  of  this  disease, 
but  in  their  broader  bearing  upon  acquired  tissue-resist- 
ance in  general. 

These  authors  found  but  little  difficulty  in  conferring 
immunity  upon  animals  that  are  otherwise  susceptible  to 
the  pathogenic  action  of  the  organisms  concerned  in  the 
production  of  this  disease,2  by  the  introduction  into  their 
tissues  of  the  products  of  growth  of  the  organisms  from 
which  the  latter  had  been  separated.  The  immunity 
thus  produced  is  seen  in  some  cases  to  last  as  long  as 
six  months ;  again  it  is  seen  to  disappear  suddenly  in 
a  way  not  to  be  explained.  It  was  seen  in  one  case 
to  be  hereditary,  probably  having  been  conferred 
upon  the  young,,  during  the  nursing-period,  through 
the  milk  of  the  mother,  as  Ehrlich3  has  shown  to 
occur  in  animals  artificially  immunized  from  abrin, 
ricin,  and  robin. 

The  energy  of  the  substance  that  has  the  power  of 
affording  immunity  was  seen  to  be  very  much  increased 
by  subjecting  it  to  temperatures  somewhat  higher  than 
that  at  which  it  was  produced  by  the  bacteria.  The 
Klemperers  found  that  if  this  substance  w^as  heated  to 

1  G.    and  F.  Klemperer  :  Berliner  klin.  Wochenschrift,   1891,  Xos. 
34  and  35. 

2  Animals  do  not,  as  a  rule,  present  the  pneumonic  changes  seen  in 
human  beings.     The  introduction  of  micrococcus  lanceolatus  into  their 
tissues  results,  in  the  case  of  susceptible  animals,  in  the  production  of 
septicaemia. 

3  Ehrlich  :  Zeitschvift  fur  Hygiene  und  Infektionskrankheiten,  1892, 
Bd.  xii.  S.  183. 


INFECTION  AND  IMMUNITY.  515 

a  temperature  of  from  41°  to  42°  C.  for  three  or  four 
days,  or  to  60°  C.  for  from  one  to  two  hours,  its  intra- 
venous injection  was  followed  by  complete  immunity 
in  from  three  to  four  days ;  whereas,  if  the  un warmed 
material  was  used,  immunity  did  not  appear  before 
fourteen  days,  and  then  only  after  the  employment  of 
relatively  large  amounts.  Moreover,  when  the  pre- 
viously heated  products  are  introduced  into  the  circu- 
lation of  the  animal  the  systemic  reaction  is  of  but 
short  duration;  but  if  the  un  warmed  substance  is 
employed,  immunity  is  manifest  only  after  the  onset 
of  considerable  elevation  of  temperature,  which  lasts 
for  a  long  time. 

In  explanation  of  these  differences  they  suggest  that, 
in  the  latter  case,  the  high  fever  that  is  seen  to  occur 
in  the  animal  may  serve  to  replace  the  warming  to 
which  the  bacterial  products  had  not  previously  been 
subjected,  and  which  is  necessary  before  they  are  in 
a  position  to  bring  about  the  condition  of  immunity. 
They  claim  that  the  bacterial  products  employed  to 
produce  immunity  in  this  case  are  not,  in  reality,  the 
immunity-affording  substance,  but  that  they  are  only 
the  agents  that  bring  about  in  the  tissues  of  the  animal 
alterations  that  result  in  the  production  of  another  body 
that  protects  the  animal.  In  support  of  this,  their 
argument  is  that  several  days  are  necessary  for  the 
production  of  immunity  by  the  introduction  into  the 
animal  of  the  bacterial  products ;  whereas,  if  the  blood- 
serum  of  this  animal,  which  is  now  protected,  be  intro- 
duced into  the  circulation  of  another  animal,  no  such 
delay  is  seen,  but  instead  the  animal  is  forthwith  pro- 
tected. In  the  former  case  the  actual  protecting  body 
had  first  to  be  manufactured  by  the  tissues;  whereas 


516  BA  CTERIOLOG  Y. 

in  the  second  it  is  already  prepared,  and  is  introduced 
as  such  into  the  second  animal. 

These  authors  found  the  serum  of  artificially  immun- 
ized animals  to  be  not  only  capable  of  rendering  other 
animals  immune,  but  to  be  possessed  of  curative  powers 
when  the  disease  was  already  in  progress.  The  serum 
of  immunized  animals  when  injected  into  the  circula- 
tion of  animals  in  which  there  was  a  body-temperature 
of  from  40.4°  to  41°  C.  reduced  this  temperature  to 
normal  (37.5°  C.)  in  twelve  consecutive  experiments 
during  the  first  twenty-four  hours  following  its  employ- 
ment. 

In  their  opinion,  the  crisis  seen  in  pneumonia  in 
human  beings  indicates  the  moment  at  which  the  pois- 
onous products,  manufactured  by  the  bacteria  located 
in  the  lungs,  are  present  in  the  circulation  in  amounts 
sufficient  to  stimulate  the  tissues  to  the  reaction  that 
results  in  the  production  of  the  antidotal  substance  that 
has  the  power  of  rendering  the  poisons  inert. 

At  the  time  of  the  crisis  in  pneumonia  the  bacteria 
themselves  are  in  no  way  affected.  They  remain  in  the 
lungs,  and  can  be  detected,  in  full  vigor  and  virulence, 
in  the  sputum  of  patients  a  long  time  after  the  disease 
is  cured.  They  have  lost  none  of  their  power  of  pro- 
ducing poisonous  products,  and  still  possess  their  orig- 
inal pathogenic  relations  toward  susceptible  animals. 
It  is  only  after  the  crisis  that  their  poisons  are  neutral- 
ized by  this  antidotal  proteid  that  has  been  produced 
by  the  cells  of  the  tissues,  and  as  this  occurs  the  sys- 
temic manifestations  gradually  disappear.  The  Klem- 
perers  claim  to  have  isolated  from  cultures  of  micro- 
coccus  lanceolatus  a  proteid  body  that  is  the  agent  con- 
cerned in  producing  the  tissue-reaction  which  results  in 


INFECTION  AND  IMMUNITY.  517 

the  formation  of  the  protecting  substance.  They  like- 
wise isolated  from  the  serum  of  immunized  animals 
a  proteid  that  possessed  the  same  powers  as  the  serum 
itself,  viz.,  of  affording  immunity  and  curing  the 
disease. 

Here,  again,  it  appears  that  the  processes  of  infection 
and  immunity  are  chemical  in  their  nature,  the  active 
poisons  of  the  invading  organisms — "  the  pneumo- 
toxins  " — being  instrumental  in  producing  the  diseased 
condition,  while  the  antidotal  or  resisting  body  of  the 
tissues  —  "the  anti-pneumotoxin "  —  is  the  agent  by 
which  the  poison  is  neutralized. 

Results  in  general  analogous  to  those  of  G.  and  F. 
Klemperer  have  also  been  obtained  by  Emmerich  and 
Fowitzky.1 

In  the  light  of  these  experiments  the  hypothesis 
advanced  by  Buchner,  that  the  establishment  of  im- 
munity is  to  be  explained  by  reactive  changes  in  the 
integral  cells  of  the  body,  receives  additional  support, 
and  when  we  consider  the  observations  of  Bitter,2  who 
found  that  in  protective  vaccinations  against  anthrax 
the  vaccines  do  not  disseminate  themselves  through 
the  body,  as  is  the  case  when  the  virulent  organisms 
are  introduced,  but  remain  at  the  site  of  inoculation, 
and  from  this  point  produce,  by  the  absorption  of  their 
chemical  products,  the  systemic  changes  through  which 
the  animal  is  protected  against  subsequent  infection  by 
the  virulent  organisms,  we  feel  justified  in  concluding 
that  the  weight  of  evidence  is  strongly  in  favor  of  this 
view. 

1  Emmerich  and  Fowitzky :  Miinchener  med.  Wocbenschrift,  1891, 
No.  32. 

2  Bitter :  Zeitschrift  fur  Hygiene,  1887,  Bd.  iv. 


518  BACTERIOLOGY. 

The  most  recent  interpretation  of  this  phenomenon 
is  that  proposed  by  Ehrlich  in  1897.1  It  is  generally 
known  as  his  "  side  chain  "  or  "  lateral  bond  "  (Seiten- 
kette)  theory  of  immunity.  It  is  one  of  the  most 
attractive  of  all  the  hypotheses  that  have  been  advanced 
in  explanation  of  this  phenomenon,  and  is  in  many 
ways  the  most  satisfactory. 

Its  important  features  comprise  the  acceptance  of 
Weigert's  doctrine  concerning  the  modus  operandi  of 
physiological  tissue-equilibrium  and  repair;  and  also 
the  assumption  of  a  specific  combining  relation,  or 
affinity,  between  toxic  substances  and  the  cells  of  par- 
ticular tissues. 

At  the  meeting  of  German  Naturalists  and  Physicians 
held  at  Frankfort-on-the-Main,  in  1896,  Weigert2  ad- 
vanced an  hypothesis  the  essential  features  of  which 
are  that  physiological  structure  and  function  depend 
upon  the  equilibrium  of  the  tissues  maintained  by  virtue 
of  mutual  restraint  between  its  component  cells  ;  that  de- 
struction of  a  single  integer  or  group  of  integers  of  a  tissue 
or  a  cell  removes  a  corresponding  amount  of  restraint  at 
the  point  injured,  and  therefore  destroys  equilibrium  and 
permits  of  the  abnormal  exhibition  of  bioplastic  ener- 
gies on  the  part  of  the  remaining  uninjured  components, 
which  activity  may  be  viewed  as  a  compensating  hyper- 
plasia ;  that  hyperplasia  is  not  therefore  the  direct 
result  of  external  irritation,  and  cannot  be,  since  the 
destructive  action  of  the  irritant  is  confined  to  the  cells 
or  integers  of  cells  that  it  destroys,  but  occurs  rather 

1  Ehrlich  :  Klinisches  Jahrbuch,  1897,  Bd.  vi.  Heft  2,  S.  309. 

2  Weigert,  Carl :  "Neue  Fragestellungen  in  der  pathologischen  Anato- 
mie,"  Verhandlungen  der  Ges.  deutscher  Naturforscher  und  Aerzte, 
1896,  S.  121. 


INFECTION  AND  IMMUNITY.  519 

indirectly  as  a  function  of  the  surrounding  uninjured 
tissues  that  have  been  excited  to  bioplastic  activity 
through  the  removal  of  .restraint  that  had  hitherto  been 
exerted  by  the  cells  destroyed  by  the  irritant;  and, 
finally,  when  such  bioplastic  activity  is  called  into  play 
there  is  always  Ai/percompensation — i.  e.y  there  is  more 
plastic  material  generated  than  is  necessary  to  com- 
pensate for  the  loss.  Ehrlich  applies  this  idea  to  the 
individual  cell,  which  he  conceives  to  be  a  complex 
molecule,  comprising  a  primary  central  nucleus  to  which 
are  attached  by  side  chains  its  secondary  atom-groups 
having  various  physiological  functions,  in  much  the 
same  way  that  our  conception  of  the  reaction-structure 
of  complex  organic  chemical  compounds  is  represented 
graphically.  Injury  to  one  or  more  of  these  physio- 
logically essential  atom-groups  results,  according  to  the 
view  of  Weigert,  in  disturbance  of  the  cell-equilibrium 
and  consequent  effort  on  the  part  of  the  surrounding 
atom-groups  at  compensatory  repair.  With  this  libera- 
tion of  bioplastic  energy  there  is  more  plastic  material 
generated  than  is  necessary  for  the  repair  of  the  injury. 
The  excess  of  this  material  finds  its  way  into  the  blood 
and,  as  we  shall  presently  see,  is  regarded  by  Ehrlich 
as  the  real  antitoxic  substance.  Assuming  a  specific 
combining  relation  between  toxic  substances  and  par- 
ticular cells  or  secondary  atom-groups  of  cells — and 
there  are  now  some  experimental  grounds  for  this 
assumption  l — it  is  evident  that  the  combination  between 
the  intoxicant  and  the  particular  atom-group  for  which 
it  has  a  specific  affinity  is  indirectly  the  cause  of  com- 

1  See  Wassennann  und  Takaki :  "  Ueber  tetanus  antitoxische  Eigen- 
schaften  des  normalen  Centralnervensystems,"  Berliner  klin.  Wocheu- 
schrift,  1898,  No.  1,  S.  5. 


520  BACTERIOLOGY. 

pensatory  bioplastic  activity  on  the  part  of  similar 
surrounding  atom-groups  that  have  not  been  destroyed 
through  combining  with  the  poison.  This  results,  as 
we  learned  above,  in  hypercompensation,  the  excess  of 
plastic  material  being  disengaged  from  the  parent-cell 
and  thrown  free  into  the  circulating  fluids,  there  to 
combine  directly  with  the  same  intoxicant  should  it 
subsequently  gain  access  to  the  animal.  This  excess 
of  plastic  material  thrown  into  the  circulation  com- 
bines, according  to  Ehrlich,1  directly  with  the  in- 
toxicant to  form  physiologically  inactive  toxin— anti- 
toxin compounds  (see  page  497),  and  can  therefore  be 
reasonably  regarded  as  the  antitoxic  material  of  arti- 
ficially immune  animals.  That  portion  of  the  cell 
which  has  the  specific  function  of  neutralizing,  by 
combining  with,  the  intoxicant  Ehrlich  designates  as 
the  "toxophoric  side  chain." 

Numerous  observations  indicate  the  probability  of  there 
being  present  in  the  blood  of  animals  several  bodies  hav- 
ing totally  different  relations  to  bacteria  and  their  prod- 
ucts, according  to  the  conditions  under  which  they  exist. 
First  there  is  present  in  the  blood-serum  of  practically 
all  animals  the  normal  defensive  "  alexins "  already 
mentioned.  Second,  the  antitoxins  that  are  found  in 
the  blood  of  animals  artificially  immunized  from  special 
sorts  of  infection  and  intoxication,  as  well  as  occasion- 
ally in  the  blood  and  tissues  of  normal  animals,  the 
functions  of  which  are  susceptible  of  demonstration 
outside  the  body  as  well  as  within  the  tissues  of  the 
living  animal.  Third,  a  body  possessed  of  disintegrating, 
bacteriolytic  powers — i.  e.,  having  the  property  of 

1  Ehrlich:"   Zur    Kenntniss  der  Antitoxinwirkung,"    Fortschritte 
der  Medicin,  1897,  Bd.  xv.  No.  2. 


INFECTION  AND  IMMUNITY.  521 

actually  breaking  bacteria  to  pieces,  so  that  the  phe- 
nomenon may  be  observed  under  the  microscope.  This 
phenomenon  is  especially  to  be  seen  within  the  peri- 
toneum of  guinea-pigs  that  have  been  rendered  immune 
from  Asiatic  cholera  and  from  the  typhoid  and  colon 
infections  or  intoxications.1  It  is  rarely  or  never  seen, 
outside  the  body,  and  it  is  not  to  be  confounded  with 
the  ordinary  bactericidal  function  of  the  alexins  that  is 
demonstrable  in  most  normal  serums.  Fourth,  a  body, 
the  so-called  "  agglutinin  "  (Gruber),  that  was  considered 
by  Widal  to  represent  a  "  reaction  of  infection,"  and 
not  of  immunity ;  though  at  this  time  its  presence  is 
generally  supposed  to  indicate  an  effort  on  the  part  of 
the  body  to  resist  infection.  The  presence  of  this  body 
in  a  serum  is  announced  by  its  peculiar  influence  on  the 
activity  and  arrangement  of  particular  bacteria  with 
which  it  is  brought  in  contact.  In  the  case  of  typhoid 
fever  in  man,  for  instance,  the  serum  obtained  during 
the  early  and  middle  stages  of  the  disease,  when  mixed 
with  fluid  cultures  or  suspensions  of  the  typhoid  bacil- 
lus, causes  the  bacilli  to  lose  their  motility  and  to  con- 
gregate (agglutinate)  in  masses  and  clumps,  a  condi- 
tion never  seen  in  normal  cultures  of  this  organism, 
and  practically  never  observed  when  normal  serum  is 
employed.  There  are  evidences  of  the  presence  of 
"agglutinin"  in  certain  of  the  antitoxic  serums  from 
artificially  immunized  animals,  viz.,  that  of  animals  im- 
mune from  cholera,  pyocyaueus,  typhoid,  and  colon  in- 
fections.  So  far  as  experience  has  gone,  this  agglutinat- 
ing property  is  manifested  in  the  great  majority  of  cases 
only  upon  the  particular  organisms  from  which  the 
animal  is  protected  ;  that  is  to  say,  the  relation  is  specific. 
1  It  is  generally  known  as  Pfeifler's  phenomenon. 


522  BACTERIOLOGY. 

In  view  of  the  fact  that  its  presence  has  been  regarded 
as  indicative  of  a  reaction  of  infection,  its  detection  in 
the  blood  of  immune  animals  may  at  first  sight  appear 
paradoxical.  We  should  not  lose  sight  of  the  fact, 
however,  that  it  is  assumed  to  be  totally  distinct  from 
the  other  substances  concerned  in  immunity,  and  its 
presence  in  immune  animals  may,  therefore,  be  reason- 
ably explained  as  a  more  or  less  permanent  result  of  the 
"  reactions  of  infection  "  that  were  coincident  with  the 
primary  injections  into  the  animal  of  infective  or  intoxi- 
cating matters  necessary  to  the  establishment  of  the 
condition  of  immunity  ;  nor  should  we  in  this  connection 
lose  sight  of  the  fact  that  its  presence  is  constantly  to 
be  demonstrated  in  typical  cases  of  typhoid  fever,  for 
instance,  that  terminate  fatally,  and  that  have  exhibited 
little  or  no  clinical  signs  of  resistance  at  any  time  dur- 
ing their  course. 

The  experiments  that  have  been  cited  afford  but  an 
imperfect  idea  of  the  enormous  amount  of  work  that 
has  been  done  upon  the  manifold  phases  of  these  im- 
portant subjects ;  they  may,  however,  serve  to  indicate 
the  direction  in  which  the  lines  of  research  have  been 
laid.  As  a  result  of  such  investigations,  our  knowledge 
upon  infection  and  immunity  may  at  present  be  sum- 
marized as  follows : 

1.  That  infection  may  be  considered  as  a  contest  be- 
tween bacteria  and  living  tissues,  conducted  on  the  part 
of  the  former  by  means  of  the  poisonous  products  of 
their  growth,  and  resisted  by  the  latter  through   the 
agency  of  proteid  bodies  normally  present  in  and  gen- 
erated by  their  integral  cells. 

2.  That  when  infection  occurs  it  may  be  explained 


INFECTION  AND  IMMUNITY.  523 

either  by  the  excess  of  vigor  of  the  bacterial  products 
over  the  antidotal  or  protective  proteids  produced  by 
the  tissues,  or  to  some  cause  that  has  interfered  with  the 
normal  activity  and  production  of  these  bodies. 

3.  That  in  the  serum  of  the  normal  circulating  blood 
of  many  animals  there  exists  a  substance  that  is  cap- 
able, outside   of  the  body,  of  rendering  inert  certain 
bacteria   which,   if  introduced   into   the   body   of  the 
animal,  would  prove  infective. 

4.  That  immunity  is  most  frequently  seen  to  follow 
the  introduction  into  the  body  of  the  products  of  growth 
of  bacteria  that  in  some  way  or  other  have  been  modi- 
fied.     This  modification  may  be   artificially  produced 
in  the  products  themselves  of  virulent  organisms,  and 
then  introduced  into  the  tissues  of  the  animal ;  or  the 
virulent  bacteria  may  be  so  treated  that  they  are  no 
longer  of  full   virulence,   and   when   introduced   into 
the   body   of  the   animal   will   produce   poisons   of  a 
much  less  vigorous  nature  than  would  otherwise  be  the 
case. 

5.  That  immunity  following  the  introduction  of  bac- 
terial products  into  the  tissues  is  not  in  all  cases  the 
result  of  the  continuous  presence  of  these  substances, 
per  se,  in  the  tissues,  or  of  a  tolerance  acquired  by  the 
tissues  to  them ;  but  is  probably,  in  certain  instances, 
due  to  the  formation  in  the  tissues  of  another  body  or 
other   bodies   that  act  as   protecting  antidotes  to  the 
poisonous  products  of  invading  organisms. 

6.  That  this  protecting  proteid  which  is  generated  by 
the  cells  of  the  tissues  need  not  of  necessity  be  antag- 
onistic to  the  life  of  the  invading  organisms  themselves, 
but  in  some  cases  must  be  looked  upon  more  as  an  anti- 
dote to  their  poisonous  products. 


524  BACTERIOLOGY. 

7.  The  immunity,  as  conceived  by  Ehrlich,  may  be 
either  "active"  or  "passive."     According  to  this  in- 
terpretation, it  is  "  active  "  when  resulting  from  an  or- 
dinary non-fatal  attack  of  infectious  disease ;  or  from  a 
mild  attack  of  infection  purposely  induced  through  the 
use  of  living  vaccines ;  or  from  the  gradual  introduc- 
tion of  toxins  into  the  tissues  until  a  marked  stage 
of  tolerance  is  reached.     It  is  "  passive  "  when  occur- 
ring as  a  result  of  the  direct  transference  of  the  per- 
fected  immunizing   substance   from   an   immune   to  a 
susceptible  animal,  as  by  the  injection  of  blood-serum 
from  the  former  into  the  latter.     "  Passive  immunity  " 
is,  in  most  cases,  conferred  at  once,  without  the  delay 
incidental  to  the  usual  modes  of  establishing  "active 
immunity."     As  a  rule,  "  active  "  is  more  lasting  than 
"  passive  "  immunity. 

8.  That  phagocytosis,  though  frequently  observed,  is 
not  essential  to  the  establishment  of  immunity,  but  is 
more  probably  a  secondary  process,  the  bacteria  being 
taken  up  by  the  leucocytes  only  after  having  been  modi- 
fied in  virulence  through  the  normal  germicidal  activity 
of  the  serum  of  the  blood  and  of  other  fluids  in  the 
body.     It  is,  however,  probable  that  the  living  leuco- 
cytes secrete  and  add  to  the  circulating  fluids  certain 
substances  that  are  important  to  the  establishment  of 
immunity. 

9.  That  of  the  hypotheses  advanced  in  explanation 
of  acquired  immunity,  the  one  worthy  of  greatest  con- 
fidence is  that  which  assumes  immunity  to  be  due  to 
reactive  changes  on  the  part  of  the  tissues  that  result 
in  the  formation  in  these  tissues  of  antitoxic  substances 
capable  of  neutralizing   the   poisons  produced   by  the 
bacteria  against  which  the  animal  has  been  immunized ; 


INFECTION  AND  IMMUNITY.  525 

though  in  certain  cases  the  establishment  of  immunity 
is  accompanied  by  the  assumption  of  germicidal  as  well 
as  antitoxic  properties  by  the  fluids  and  tissues,  and  in 
a  few  instances  the  germicidal  is  more  pronounced  than 
the  antitoxic  function. 


CHAPTER  XXVII. 

Bacteriological  study  of  water— Methods  employed— Precautions  to  be 
observed — Apparatus  used,  and  methods  of  using  them — Methods 
of  investigating  air  and  soil. 

BACTERIOLOGICAL   STUDY   OF   WATER. 

THE  conditions  that  favor  the  epidemic  outbreak  of 
typhoid  fever,  Asiatic  cholera,  and  other  maladies  of 
which  these  may  be  taken  as  types,  have  served  as  a 
subject  for  discussion  by  sanitarians  for  a  long  time. 

Of  the  hypotheses  that  have  been  advanced  in  ex- 
planation of  the  existence  and  dissemination  of  these 
diseases,  two  stand  pre-eminent  and  are  worthy  of  con- 
sideration. They  are  the  "  ground- water "  theory  of 
von  Pettenkofer  and  his  pupils,  and  the  "  drinking- 
water  "  theory  of  the  school  of  bacteriologists  of  which 
Koch  stands  at  the  head. 

The  adherents  to  the  "  ground- water  "  view  explain 
the  occurrence  of  these  diseases  in  epidemic  form  through 
alterations  in  the  soil  resulting  from  fluctuations  in  the 
level  of  the  soil-water;  and  assign  to  drinking-water 
either  a  very  insignificant  role,  or,  as  is  most  frequently 
the  case,  ignore  it  entirely.  On  the  other  hand,  those 
who  have  been  instrumental  in  developing  the  drinking- 
water  hypothesis  claim  that  alterations  in  the  soil  play 
little  or  no  part  in  favoring  the  outbreak  of  these  dis- 
eases ;  but  that,  as  a  rule,  they  appear  as  a  result  of 
direct  infection,  through  the  use  of  waters  contaminated 

526 


BACTERIOLOGICAL  STUDY  OF   WATER.        527 

with  materials  containing  the  specific  organisms  known 
to  cause  such  diseases. 

As  a  result  of  numerous  observations  by  the  disciples 
of  both  schools,  the  evidence  is  now  greatly  in  favor  of  the 
opinion  that  polluted  water  is  primarily  the  underlying 
cause  of  these  epidemics,  and  this  too,  very  often,  when 
the  state  of  the  soil-water,  in  the  light  of  the  "  ground- 
water"  hypothesis,  is  just  the  reverse  of  what  it  should 
be  in  order  to  render  it  answerable  for  them.  It  is 
manifest,  therefore,  that  the  careful  bacteriological  study 
of  water  intended  for  domestic  use  is  of  the  greatest 
importance,  and  should  be  a  routine  procedure  in  all 
communities  receiving  their  water-supply  from  sources 
liable  to  pollution. 

The  object  aimed  at  in  such  investigations  should  be 
to  determine  the  number  and  kind  of  bacteria  con- 
stantly present  in  the  water — for  all  waters,  except 
deep  ground- wrater,  contain  bacteria  ;  if  sudden  fluctua- 
tions in  the  number  and  kind  of  bacteria  occur  in 
these  waters,  and  if  so,  to  what  they  are  due ;  and 
finally,  and  most  important,  Does  the  water  contain 
constantly,  or  at  irregular  periods,  bacteria  that  can  be 
traced  to  human  excrement,  not  of  necessity  pathogenic 
varieties,  but  bacteria  that  are  known  to  be  present 
normally  in  the  intestinal  canal?  For  if  conditions 
are  continuously  favorable  to  pollution  of  the  water 
by  the  normal  constituents  of  the  intestinal  canal,  the 
same  conditions  would  allow  of  the  occasional  pollution 
of  such  water  by  infective  matters  from  the  bowels  of 
persons  suffering  from  specific  disease  of  the  intestines. 

In  considering  water  from  a  bacteriological  stand- 
point it  must  always  be  borne  in  mind  that  com- 
parisons with  fixed  standards  are  not  of  much  value, 


528  BACTERIOLOGY. 

for  just  as  normal  waters  from  different  sources  are 
seen  to  present  variations  in  their  chemical  composition, 
without  being  unfit  for  use,  so  may  the  relative  number 
and  variety  of  species  of  bacteria  in  water  from  one 
source  be  always  greater  or  smaller  than  in  that  from 
another,  and  yet  no  difference  may  be  seen  to  result 
from  their  employment.  For  this  reason  systematic 
study  of  any  water,  from  this  point  of  view,  should 
begin  with  the  establishment  of  what  may  be  called  its 
normal  mean  number  of  bacteria,  as  well  as  the  charac- 
ter of  the  prevailing  species ;  and  in  order  to  do  this 
the  investigations  must  cover  a  long  period  of  time 
through  all  the  seasonal  variations  of  weather.  From 
data  obtained  in  this  way  it  may  be  possible  without 
analysis  to  predict  approximately  at  any  season  the 
bacteriological  condition  of  the  water  studied.  Marked 
deviations  from  these  "  means,"  either  in  the  quantity 
or  quality  of  the  organisms  present,  can  then  be  con- 
sidered as  indicative  of  the  existence  of  some  unusual, 
disturbing  element,  the  nature  of  which  should  be 
investigated.  It  is  impossible  to  formulate  an  opinion 
of  much  value  from  either  a  single  chemical  or  bac- 
teriological analysis  of  a  water,  or  from  both  together 
in  many  cases ;  for  the  results  thus  obtained  indicate 
only  the  condition  of  the  water  at  the  time  the  sample 
was  procured,  and  give  no  indication  as  to  whether  it 
differed  at  that  time  from  its  usual  condition,  or  from 
the  normal  condition  of  the  waters  of  the  immediate 
neighborhood. 

The  interpretation  of  the  results  of  both  chemical 
and  bacteriological  analyses  of  a  sample  of  water  ac- 
quires its  full  value  only  through  comparison,  either 
with 


BACTERIOLOGICAL  STUDY  OF  WATER.        529 

water,  or  with  the  results  of  simultaneous  analyses  of 
a  number  of  samples  from  other  sources  of  supply  of 
the  locality. 

The  aid  of  the  bacteriologist  is  frequently  sought  in 
connection  with  investigations  of  waters  that  are  sup- 
posed to  be  concerned  in  the  production  of  disease,  par- 
ticularly typhoid  fever,  either  in  isolated  cases  or  in 
\\idcspread  epidemic  outbreaks,  and  in  these  cases  both 
the  bacteriologist  and  the  person  employing  his  services 
are  cautioned  against  being  too  sanguine  of  positive 
results,  for  in  the  vast  majority  of  instances  reliable 
bacteriologists  fail  to  detect  in  these  waters  the  bacillus 
that  is  the  cause  of  typhoid  fever. 

Failure  to  find  the  organism  of  typhoid  fever  in 
water  by  the  usual  methods  of  analysis  does  not  by 
any  means  prove  that  it  is  not  present  or  has  not 
been  present.  The  means  ordinarily  employed  in  the 
work  admit  of  such  a  very  small  volume  of  water  being 
used  in  the  test  that  we  can  readily  understand  how 
typhoid  bacilli  might  be  present  in  moderate  numbers 
and  yet  none  be  included  in  the  drop  or  twx>  of  the 
water  taken  for  study.  The  conditions  are  not  those 
of  a  solution,  each  drop  of  which  contains  exactly  as 
much  of  the  dissolved  material  as  do  all  other  drops  of 
equal  volume ;  but  are  rather  those  of  a  suspension,  in 
every  drop  or  volume  of  which  the  number  of  sus- 
pended particles  is  liable  to  the  greatest  degree  of 
variation.  Furthermore,  there  are  other  reasons  that 
would,  a  priwij  preclude  our  expecting  to  find  the 
typhoid  bacilli  in  water  in  which  we  may  have  reason 
to  believe  they  had  been  deposited,  viz.,  attention  is 
not  usually  directed  to  the  water  until  the  disease 
has  become  conspicuous,  usually  in  from  two  to  four 

34 


530  BACTERIOLOGY. 

weeks  after  the  pollution  probably  occurred.  These 
intervals  of  time  are  ordinarily  sufficient  for  the  deli- 
cate, non-resistant  bacillus  of  typhoid  fever  to  succumb 
to  the  unfavorable  conditions  under  which  it  finds  itself 
in  water.  By  unfavorable  conditions  are  meant  the 
absence  of  suitable  nutrition ;  unfavorable  temperature ; 
probably  the  antagonistic  influence  of  more  hardy 
saprophytic  bacteria,  particularly  the  so-called  "  water- 
bacteria/7  and  of  more  highly  organized  water-plants ; 
the  effect  of  precipitation  and  of  sedimentation ;  and, 
of  great  importance,  the  disinfecting  action  of  direct 
sunlight. 

Though  the  positive  demonstration  of  typhoid  bacilli 
in  drinking-water  by  bacteriological  methods  is  of  ex- 
treme rarity,  it  must  not  be  concluded  that  bacteriological 
analyses  of  suspicious  waters  shed  no  light  upon  the  exist- 
ence of  pollution  and  the  suitability  or  non-suitability 
of  the  water  for  drinking-purposes. 

In  the  normal  intestinal  tract  of  all  human  beings 
and  of  many  other  mammals,  as  well  as  associated  with 
the  specific  disease-producing  bacterium  in  the  intes- 
tines of  typhoid-fever  patients,  is  an  organism  that  is 
frequently  found  in  polluted  drinking-waters,  and 
whose  presence  is  proof  positive  of  pollution  by  either 
normal  or  diseased  intestinal  contents ;  and  though 
efforts  may  result  in  failure  to  detect  the  specific  bacil- 
lus of  typhoid  fever,  the  finding  of  the  other  organism, 
bacterium  coli  commune,  justifies  one  in  expressing  the 
opinion  that  the  water  under  consideration  has  been 
polluted  by  intestinal  evacuations  from  either  human 
beings  or  animals.  Waters  so  exposed  as  to  be  liable  to 
such  pollution  should  never  be  considered  as  other  than 
a  continuous  source  of  danger  to  those  using  them. 


BACTERIOLOGICAL  STUDY  OF  WATER.        531 

Another  point  to  be  remembered  is  in  connection  with 
chlorine  as  an  indicator  of  contamination  by  human 
excrement.  It  is  commonly  taught  that  an  excessive 
amount  of  chlorine  in  water  points  to  contamination  by 
human  excreta.  This  may  or  may  not  be  true,  accord- 
ing to  circumstances.  A  high  proportion  of  this  element 
in  a  sample  of  water  from  a  locality,  the  surrounding 
waters  of  which  are  poor  in  chlorine,  is  unquestionably 
a  suspicious  indication  ;  but  in  a  district  close  to  the  sea 
or  near  salt-deposits,  for  instance,  where  the  proportion 
of  chlorine  (as  chlorides)  in  the  water  is  generally 
high,  the  value  of  the  indications  thus  afforded  is  very 
much  diminished  unless  the  amount  found  in  the  sample 
under  examination  greatly  exceeds  the  normal  "  mean," 
previously  determined,  for  the  amount  of  chlorine  in 
the  waters  of  the  neighborhood. 

A  striking  example  of  the  latter  condition  occurred 
in  the  experience  of  the  writer  while  inspecting  a 
group  of  water-supplies  on  the  east  coast  of  Florida. 
In  each  instance  the  water  was  obtained  from  properly 
protected  artesian  wells,  ranging  from  200  to  400 
feet  deep,  and  located  within  a  few  hundred  yards 
of  the  sea.  The  first  sample  subjected  to  chemical 
analysis  revealed  such  an  unusually  high  proportion 
of  chlorine  that,  had  this  sample  alone  been  con- 
sidered, the  opinion  that  it  was  polluted  by  human 
excreta  might  have  been  advanced.  To  prevent  such 
an  error  samples  of  water  from  a  number  of  wells  in 
the  neighborhood  were  examined,  and  they  were  all 
found  to  contain  from  ten  to  twelve  times  the  amount 
of  chlorine  that  ordinarily  appears  in  inland  waters,  the 
excess  being  evidently  due  to  leakage  through  the  soil 
into  the  wells  of  water  from  the  sea.  In  short,  the 


532  BACTERIOLOGY. 

presence  of  an  excess  of  chlorine  in  water,  while  often 
indicating  pollution  from  human  evacuations,  may 
nevertheless,  sometimes  arise  from  other  sources ;  but 
the  presence  in  water  of  bacteria  normally  found  in  the 
intestinal  canal  can  manifestly  admit  of  but  one  inter- 
pretation, viz.,  that  faecal  matters  from  either  man  or 
animals  have  at  some  time  been  deposited  in  this  water, 
and  that  while  no  specific  disease-producing  organisms 
may  have  been  detected,  still  waters  in  which  such  pol- 
lutions are  possible  are  a  constant  menace  to  the  health 
of  those  who  use  them  for  domestic  purposes. 

A  sudden  variation  from  the  normal,  mean  number 
of  bacteria,  or  from  the  normal  chemical  composi- 
tion of  a  water,  calls  at  once  for  a  thorough  in- 
spection of  the  supply,  while  at  the  same  time  the 
organisms  present  are  to  be  subjected  to  the  most  care- 
ful study.  In  many  instances,  even  after  the  most 
thorough  bacteriological  and  chemical  study  of  a  sus- 
picious watefr,  one  is  forced  to  admit  that  information 
of  but  limited  usefulness  has  been  obtained  through  the 
employment  of  such  analytical  methods.  In  these 
cases  too  much  stress  cannot  be  laid  upon  the  im- 
portance of  a  systematic  inspection  of  the  supply,  and 
its  relation  to  sources  of  pollution.  Optical  evidence 
of  more  or  less  dangerous  contamination  may  often  be 
obtained  when  laboratory  methods  fail  to  detect  them. 
The  reasons  for  such  failure,  in  addition  to  those  already 
given,  are  obvious — the  polluting  matters  are  often  so 
diluted  by  the  large  mass  of  water  into  which  they  find 
their  way  as  to  be  beyond  recognition  by  the  tests 
usually  employed  in  such  work,  and  still  be  present  in 
amounts  sufficient  to  originate  disease. 

THE   QUALITATIVE    BACTERIOLOGICAL   ANALYSIS 


BACTERIOLOGICAL  STUDY  OF  WATER.        533 

OF  WATER. — The  qualitative  bacteriological  analysis 
of  water  entails  much  labor,  as  it  requires  not  only 
that  all  the  different  species  of  organisms  found  in  the 
water  should  be  isolated,  but  that  each  representative 
should  be  subjected  to  systematic  study,  and  its  patho- 
genic or  non-pathogenic  properties  determined. 

For  this  purpose  a  knowledge  of  the  methods  for  the 
isolation  of  individual  species  which  have  already  been 
described,  and  of  the  means  of  studying  these  species 
when  isolated,  is  indispensable. 

For  this  analysis  certain  precautions  essential  to 
accuracy  are  always  to  be  observed. 

The  sample  is  to  be  collected  under  the  most  rigid 
precautions  that  will  exclude  organisms  from  sources 
other  than  that  under  consideration.  If  drawn  from  a 
spigot,  it  should  never  be  collected  until  the  water  has 
been  flowing  for  fifteen  to  twenty  minutes  in  a  full 
stream.  If  obtained  from  a  stream  or  a  spring,  it 
should  be  collected,  not  from  the  surface,  but  rather 
from  about  one  foot  beneath  the  surface. 

It  should  always  be  collected  in  vessels  which  have 
previously  been  thoroughly  freed  from  all  dirt  and 
organic  particles,  and  then  sterilized ;  and  the  plates 
should  be  made  as  quickly  as  possible  after  collecting 
the  sample. 

When  circumstances  permit,  all  water  analyses  should 
be  made  on  the  spot  where  the  sample  is  taken,  as  it 
is  known  that  during  transportation,  unless  the  samples 
are  kept  packed  in  ice,  a  multiplication  of  the  organ- 
isms contained  in  it  always  occurs. 

For  the  purpose  of  qualitative  analysis  it  is  necessary 
that  a  small  portion  of  the  water — one,  two,  three,  five 
drops — should  first  be  employed  for  making  the  plates. 


534  BACTERIOLOGY. 

In  this  way  one  can  form  an  idea  as  to  the  approximate 
number  of  organisms  in  the  water,  and  can,  in  conse- 
quence, determine  the  amount  of  water  best  suited  for 
the  plates.  Duplicate  plates  are  always  to  be  made — 
one  set  upon  agar-agar,  which  are  to  be  kept  in  the 
incubator  at  body-temperature,  and  one  set  upon  gelatin, 
to  be  kept  at  from  18°  to  20°  C. 

As  soon  as  colonies  have  developed  the  plates  are  to 
be  carefully  compared  and  studied.  It  is  to  be  noted 
if  any  difference  in  the  appearance  of  the  organisms  on 
corresponding  plates  exists,  and  if  so,  to  what  it  is  due. 
It  is  to  be  particularly  noted  which  plates  contain  the 
greater  number  of  colonies,  those  kept  at  the  higher  or 
those  at  the  lower  temperature.  In  this  way  the  tem- 
perature best  suited  for  the  growth  of  the  majority  of 
these  organisms  may  be  determined.  As  a  rule,  the 
greater  number  of  colonies  appear  upon  the  gelatin 
plates  kept  at  18°  to  20°  C. ;  and  from  this  it  would 
seem  that  many  of  the  normal  water-bacteria  do  not 
find  the  higher  temperature  so  favorable  to  their  de- 
velopment as  do  the  organisms  not  naturally  present  iu 
water,  particularly  the  pathogenic  varieties.  From  these 
plates  the  different  species  are  to  be  isolated  in  pure 
culture,  the  morphological  and  cultural  characteristics 
determined,  and  finally,  by  tests  upon  animals,  it  is  to 
be  decided  if  any  of  them  possess  disease-producing 
properties. 

NOTE. — What  use  should  be  made  of  this  observa- 
tion in  examining  water  for  the  presence  of  pathogenic 
bacteria  ? 

The  waters  most  frequently  studied  from  the  quali- 
tative bacteriological  standpoint  are  those  suspected 


BACTERIOLOGICAL  STUDY  OF    WATER,       535 

of  containing  specific  pathogenic  bacteria — /.  e.y  waters 
polluted  with  sewage  and  witli  human  excreta  that 
are  believed  to  be  the  source  of  infection  of  typhoid 
fever,  or,  less  frequently,  of  Asiatic  cholera.  In  the 
investigations  of  such  waters  there  are  several  points  of 
which  we  should  never  lose  sight,  viz.,  unless  the 
water  is  under  continuous  study  there  is  only  a  chance 
of  detecting  the  specific  pathogenic  species,  for,  as  a  rule, 
the  dangerous  pollution  occurs  either  but  once  or  is 
intermittent,  so  that  even  in  the  case  of  exposed  streams 
there  are  periods  when  no  specifically  dangerous  con- 
tamination may  be  in  operation.  As  stated  above,  atten- 
tion is  commonly  called  to  the  water  when  the  disease, 
presumably  caused  by  its  use,  is  fully  developed,  and 
this  is  often  days  or  weeks  after  the  infection  of  the 
stream  really  occurred.  By  an  analysis  made  at  this 
time  one  could  scarcely  hope  to  detect  the  specific  organ- 
isms that  had  caused  the  disease.  The  organisms  sought 
for  may  have  been  present  in  the  water  and  may  have 
infected  the  users,  and  yet  have  disappeared  by  the  time 
the  sample  taken  for  analysis  was  collected. 

When  present  in  polluted  waters  pathogenic  bacteria 
are  always  vastly  in  the  minority.  Were  they  con- 
stantly present  in  large  numbers  infection  among  the 
users  of  such  waters  would  be  more  frequent  and  more 
widespread  than  is  commonly  the  case.  They  may  be 
present  in  a  water-supply  in  small  numbers ;  they  may 
even  be  in  the  sample  supplied  for  analysis,  and  yet  es- 
cape detection  if  only  the  ordinary  direct  plate  method 
of  isolation  be  used. 

From  these  considerations  it  is  obvious  that  before 
attempts  are  made  to  isolate  the  various  species  directly 
from  a  suspicious  sample  of  water  it  is  advisable  to 


536  BACTERIOLOGY. 

subject  it  to  some  method  of  treatment  that  will  aid  in 
separating  the  few  specific  pathogenic  from  the  numer- 
ous common  saprophytic  species.  For  this  purpose 
numerous  methods  have  been  devised.  The  most  use- 
ful of  these  aim  to  favor  the  rapid  multiplication  of 
pathogenic  forms  that  may  be  present  and  to  suppress 
or  check  the  growth  of  the  ordinary  water  saprophytes. 

Attention  has  been  called  to  the  fact  that  when  ex- 
posed to  the  body-temperature  many  of  the  ordinary 
water-bacteria  develop  only  slowly  or  not  at  all,  while 
under  similar  circumstances  the  disease-producing  spe- 
cies develop  most  luxuriantly.  Advantage  has  been 
taken  of  this  observation  in  devising  methods  for  this 
particular  work,  of  which  some  of  the  following  will 
prove  serviceable : 

Collect  in  a  sterilized  flask  a  sample  of  about  100  c.c. 
of  the  water  to  be  tested,  and  add  to  this  about  25  c.c. 
of  sterilized  bouillon  of  four  times  the  usual  strength. 
This  is  then  placed  in  the  incubator  at  37°  to  38  °C., 
for  thirty-six  to  forty-eight  hours,  after  which  plates  are 
to  be  made  from  it  in  the  usual  way ;  the  results  will 
often  be  a  pure  culture  of  some  single  organism,  either 
one  of  the  intestinal  variety  or  a  closely  allied  species. 
By  a  method  analogous  to  the  latter  the  spirillum  of 
Asiatic  cholera  has  been  isolated  from  water  (see  pages 
410,  425)  ;  and  by  taking  advantage  of  the  eifect  of  ele- 
vated temperature  upon  the  bacteria  of  water  Vaughan 
has  succeeded  in  isolating  from  suspicious  waters  a 
group  of  organisms  very  closely  allied  to  the  bacillus 
of  typhoid  fever. 

Theobald  Smith  has  suggested  a  method  by  which  it 
is  easily  possible  to  isolate,  from  waters  in  which  they 
are  present,  certain  organisms  that  are  of  the  utmost 


BACTERIOLOGICAL  STUDY  OF  WATER.        537 

importance  in  influencing  our  judgment  upon  the  fitm  — 
of  the  water  for  domestic  use.  By  the  addition  of  small 
quantities — one,  two,  or  three  drops — of  the  suspicious 
water  to  fermentation-tubes  (see  article  an  Fermenta- 
tion-tube) containing  bouillon  to  which  2  per  cent,  of 
glucose  has  been  added,  and  keeping  them  at  the  tempera- 
ture of  the  body  (37°  to  38°  C.),  the  growth  of  intestinal 
bacteria  that  may  be  present  in  the  water  is  favored, 
while  that  of  the  water-organisms  is  not ;  in  consequence, 
after  from  thirty-six  to  forty-eight  hours  the  fer- 
mentation-characteristics of  most  of  these  organisms  is 
evidenced  by  the  accumulation  of  gas  in  the  closed  end 
of  the  tube.  From  these  tubes  the  growing  bacteria 
can  then  be  easily  isolated  by  the  plate  method,  and 
intestinal  bacteria  will  not  infrequently  be  found 
present. 

For  the  isolation  of  the  typhoid  bacillus,  especially  from 
water,  a  host  of  other  methods  have  been  devised.  Some 
of  these  aim,  through  the  addition  of  special  chemical 
reagents  to  the  media,  to  retard  the  development  of 
ordinary  saprophytes  without  interrupting  the  growth 
of  the  colon  and  the  typhoid  bacillus.  Most  of  these 
methods  have  proved  disappointing.  One  of  them,  that 
of  Parietti,  still  finds  favor  in  the  hands  of  some.  It 
consists  in  adding  to  the  culture-media  to  be  used  in 
the  test  varying  amounts  of  the  following  mixture : 

Phenol 5  grammes. 

Hydrochloric  acid 4 

Distilled  water 100  c.c. 

Of  this  solution  0.1,  0.2,  and  0.3  c.c.  are  added  re- 
spectively to  each  of  three  tubes  containing  10  c.c.  of 
nutritive  bouillon.  Several  such  sets  of  tubes  are  to  be 


538  BACTERIOLOGY. 

made.  To  each  are  then  added  from  1  to  3  c.c.  of  the 
water,  and  they  are  placed  in  the  incubator  at  body-tem- 
perature. It  is  said  that  whatever  development  occurs 
consists  only  of  the  typhoid  or  colon  bacillus,  or  both,  if 
they  were  present  in  the  original  sample.  They  may 
then  be  isolated  and  separated  by  the  usual  plate  method, 
or,  better  still,  through  the  application  of  the  methods 
of  Eisner,  Stoddart,  or  Hiss,  detailed  in  the  chapter 
on  bacillus  typhosus.  Personally  we  have  not  had 
much  success  with  the  Parietti  method.  The  typhoid 
bacillus  has  been  isolated  from  water  by  passing  very 
large  quantities  of  water  through  an  ordinary  Pasteur 
or  Birkfield  filter,  brushing  off  the  matters  collected 
on  the  filter  into  a  sterilized  vessel  and  examining  this 
by  plate  methods. 

It  has  occurred  to  us  that  possibly  the  employment 
of  chemical  coagulants,  such  as  alum  and  iron,  might 
prove  serviceable  for  this  purpose.  Their  action  would 
be  to  mechanically  drag  down,  in  precipitating  as  hy- 
droxides, the  suspended  bacteria  contained  in  the  fluid. 
This  precipitate  could  then  be  examined  bacteriologi- 
cally,  instead  of  the  water. 

The  difficulties  in  this  field  of  work  are  obviously 
due  to  the  suspension  of  a  very  small  number  of  the 
disease-producing  organisms  sought  for  in  large  volumes 
of  fluid,  and  the  association  with  them  of  large  numbers 
of  other  species  that  offer  a  very  great  obstacle  to  the 
successful  search  for  the  pathogenic  varieties. 

If  by  either  of  the  above  procedures  bacilli  that  bear 
any  resemblance  to  bacillus  typhosus  be  isolated,  re- 
course must  then  be  had  to  all  the  differential  tests 
detailed  in  the  chapter  on  that  organism. 

THE  QUANTITATIVE  ESTIMATION  OF  BACTERIA  IN 


BACTERIOLOGICAL  STUDY  OF  WATER.        539 

WATER. — Quantitative  analysis  requires  more  care  IP 
the  measurement  of  the  exact  volume  of  water  em- 
ployed, for  the  results  are  to  be  expressed  in  terms  of  the 
number  of  individual  organisms  to  a  definite  volume. 
The  necessity  for  making  the  plates  at  the  place  at 
which  the  sample  is  collected  is  to  be  particularly 
accentuated  in  this  analysis,  for  multiplication  of  the 
organisms  during  transit  is  so  great  that  the  results 
of  analyses  made  after  the  water  has  been  in  a  vessel 
for  a  day  or  two  are  often  very  different  from  those  that 
would  have  been  obtained  on  the  spot. 

NOTE. — Inoculate  a  tube  containing  about  ten  cubic 
centimetres  of  sterilized  distilled  or  tap  water  with  a 
very  small  quantity  of  a  solid  culture  of  some  one  of 
the  organisms  with  which  you  have  been  working, 
taking  care  that  none  of  the  culture-medium  is  intro- 
duced into  the  water-tube  and  that  the  bacteria  are 
evenly  distributed  through  it.  Make  plates  at  once, 
and  on  each  succeeding  day,  from  this  tube,  and  deter- 
mine by  counts  whether  there  is  an  increase  or  diminu- 
tion in  the  number  of  organisms — i.  e.y  if  they  are 
growing  or  dying.  Represent  the  results  graphically, 
and  it  will  be  noticed  that  in  many  cases  there  is 
during  the  first  three  or  four  days  a  multiplication, 
after  which  there  is  a  rapid  diminution ;  and,  if  the 
organism  does  not  form  spores,  usually  death  in  from 
ten  to  twelve  days.  This  is  not  true  for  all  organisms, 
but  does  hold  for  many. 

Where  it  is  not  convenient,  however,  to  make  the 
analysis  on  the  spot,  the  sample  of  water  should  be 
taken  and  packed  in  ice  and  kept  on  ice  until  the  plates 


540  BACTERIOLOGY. 

can  be  made  from  it,  which  should  in  all  cases  be  as 
soon  after  its  collection  as  possible. 

For  the  collection  of  water  for  this  purpose  a  con- 
venient vessel  is  a  glass  bulb  (Fig.  100)  or  balloon, 
which  is  easily  blown  from  glass  tubing. 

FIG.  100. 


Glass  bulb  for  collecting  samples  of  water. 

It  consists  simply  of  a  round  glass  sphere  blown  on 
the  end  of  a  glass  tube,  which  latter  is  subsequently 
drawn  out  into  a  capillary  stem  and  sealed  while  hot. 
As  it  cools,  the  contraction  of  the  air  within  the  bulb 
results  in  the  establishment  of  a  negative  pressure. 

If  the  point  of  the  stem  be  broken  off  under 
water,  the  water  flows  into  the  bulb,  because  of  the 
existence  of  the  negative  pressure  within.  The  nega- 
tive pressure  obtained  in  this  way  is  frequently  in- 
sufficient for  filling  the  bulb,  and  often  only  a  few 
drops  of  fluid  can  be  obtained.  To  obviate  this, 
bulbs  may  be  blown  and  allowed  to  cool,  but  not 
sealed.  After  a  sufficient  number  of  them  are  pre- 
pared they  are  taken,  one  at  a  time,  and  gently 
warmed  over  a  flame ;  while  still  warm  the  extremity 
of  the  stem  is  dipped  into  distilled  water  and  held 
there  until  a  few  drops  have  passed  up  into  the  bulb ; 
this  is  then  carefully  boiled,  or,  rather,  completely  vapor- 
ized, over  the  flame,  and  while  steam  is  still  escaping 
the  point  is  sealed  in  the  gas-flame.  All  air  will 
thus  have  been  replaced  by  water-vapor,  and  if  the 
point  of  the  stem  be  now  broken  off  under  water 


BACTERIOLOGICAL  STUDY  OF  WATER.        541 

the  bulb  will  fill  quickly.  It  is  not  desirable  to 
fill  them  completely,  but  rather  to  only  about  three- 
fourths  of  their  capacity,  as  when  full  it  is  difficult 
to  empty  them  without  contaminating  the  contents. 
They  are  emptied  by  gently  warming  over  a  gas  or 
alcohol  flame. 

A  number  of  bulbs  may  be  made,  sealed,  and  kept 
on  hand.  They  are  sterile  so  long  as  they  are  sealed, 
because  of  the  heat  employed  in  their  manufacture. 

When  a  sample  of  water  is  to  be  taken,  the  point 
of  a  bulb  is  simply  broken  off  with  sterilized  forceps 
under  water  and  held  there  until  the  necessary  amount 
has  been  obtained.  This  may  serve  as  a  sample  from 
\vhich  to  prepare  plates  or  Esmarch  tubes  on  the  spot ; 
or  the  tip  of  the  stem  may  be  resealed  in  the  flame 
of  an  alcohol  lamp,  the  bulb  packed  in  ice,  and  trans- 
ported in  this  condition  to  the  laboratory. 

Another  very  simple  and  useful  device  for  collecting 
water  samples  is  that  recommended  by  Kirschner.  This 
consists  of  a  piece  of  glass  tubing  of  about  5  or  6  mm. 
inside  diameter,  and  36  cm.  long,  bent  in  the  form  of 
a  U,  with  either  extremity  of  the  arms  bent  again  at 
right  angles  in  the  same  plane  and  drawn  out  to  a  point 
and  sealed.  Such  tubes  are  sterilized  in  the  flame  as  they 
are  made.  The  sample  is  collected  by  breaking  off 
both  points,  immersing  one  of  them  in  Avater  and  suck- 
ing on  the  other  until  the  tube  is  filled.  Then  both 
points  are  again  sealed  in  the  flame  and  the  tube  packed 
in  ice.  The  objection  to  this  tube  is  the  danger  of  con- 
taminating its  contents  with  saliva  during  the  act  of 
filling  by  suction,  though  this  danger  is  not  so  great 
as  might  at  first  appear,  as  we  shall  learn  in  our  efforts 
to  cultivate  bacteria  from  the  mouth-cavity. 


542  BACTERIOLOGY. 

NOTE. — Make   cover-slips   from  your   own  mouth ; 
make  plates  on  both  gelatin  and  agar-agar,  at  the  same 

time.    Compare  the  number  of  bacteria 
FIG.  101.  \  .  . 

found  by  microscopic  examination  of 

the  cover-slips  with   the  number  of 
colonies  that  develop  on  the  plates. 

For  the  collection  of  samples  from 
the  deeper  portions  of  streams,  lakes, 
etc.,  a  number  of  convenient  devices 
have  been  made.     A  very  satisfactory 
apparatus  has  been  made  for  me  by 
Messrs.  Charles  Leu  tz&  Sons,  of  Phila- 
delphia.    It  consists  of  a  metal  frame- 
work,  in  which   is   encased   a  bottle 
provided  with  a  ground-glass  stopper. 
To  the  stopper  a  spring  clamp  is  at- 
tached, and  this  in  turn  is  operated  by 
a  string,  so  that  when  the  weighted 
apparatus  is  allowed  to  sink  into  the 
stream  the  stopper  may  be  removed 
from  the  bottle  at  any  depth  by  simply 
pulling  upon  the   string.     When  the 
bottle  is  filled  with  water  the  stopper 
is  allowed  to  spring  back  into  position 
by  releasing  the  string.     The   whole 
B0"le  wIte°r!leCting  apparatus  (depicted  in  Fig.  101)  is  pro- 
vided with  a  weight  that  insures  its 
sinking,  and  a  heavy  cord  by  which  it  may  be  lowered 
and  raised.     It  should  be  sterilized  before  using.    After 
collecting  the  sample  the  bottle  should  be  wiped  dry 
with  a  sterilized  towel.     Before  removing  the  stopper 
the  mouth  of  the  bottle  should  be  rinsed  with  alcohol 


BACTERIOLOGICAL  STUDY  OF  WATER.        543 

and  heated  with  a  gas-flame,  to  prevent  contamination 
of  its  contents  by  matters  that  may  have  been  upon 
its  surface. 

In  beginning  the  quantitative  analysis  of  water  with 
which  one  is  not  acquainted  certain  preliminary  steps 
are  essential. 

It  is  necessary  to  know  approximately  the  number  of 
organisms  contained  in  any  fixed  volume,  so  as  to  deter- 
mine the  quantity  of  water  to  be  employed  for  the  plates 
or  tubes.  This  is  usually  done  by  making  preliminary 
plates  from  one  drop,  two  drops,  0.25  c.c.,  0.5  c.c.,  and 
1  c.c.  of  the  water.  After  each  plate  has  been  labelled 
with  the  amount  of  water  used  in  making  it,  it  is  placed 
aside  for  development.  When  this  has  occurred  one 
selects  the  plate  upon  which  the  colonies  are  only  mod- 
erate in  number — about  200  to  300  colonies  presenting 
— and  employs  in  the  subsequent  analysis  the  same 
amount  of  water  that  was  used  in  making  this  plate. 

If  the  original  water  contained  so  many  organisms 
that  there  developed  on  a  plate  or  tube  made  with  one 
drop  too  many  colonies  to  be  easily  counted,  then  the 
sample  must  be  diluted  with  one,  ten,  twenty-five,  fifty, 
or  one  hundred  volumes,  as  the  case  may  require,  of 
sterilized  distilled  water.  This  dilution  must  be  accu- 
rate, and  its  exact  extent  noted,  so  that  subsequently  the 
number  of  organisms  per  volume  in  the  original  wrater 
may  be  calculated. 

The  use  of  a  drop  is  not  sufficiently  accurate.  The 
dilution  should  therefore  always  be  to  a  degree  that  will 
admit  of  the  employment  of  a  volume  of  water  that 
may  be  exactly  measured,  0.25  and  0.5  c.c.  being  the 
amounts  most  convenient  for  use. 

Duplicate  plates  should   always   be   made,  and  the 


544  BACTERIOLOGY. 

mean  of  the  number  of  colonies  that  develop  upon 
them  taken  as  the  basis  from  which  to  calculate  the 
number  of  organisms  per  volume  in  the  original  water. 

For  example:  from  a  sample  of  water  0.25  c.c.  is 
added  to  a  tube  of  liquefied  gelatin,  carefully  mixed  and 
poured  as  a  plate.  When  development  occurs  the 
number  of  colonies  are  too  numerous  to  be  accurately 
counted.  One  cubic  centimetre  of  the  original  water 
is  then  to  have  added  to  it,  under  precautions  that  pre- 
vent contamination  from  without,  99  c.c.  of  sterilized 
distilled  water — that  is,  we  have  now  a  dilution  of 
1  :  100.  Again,  0.25  c.c.  of  this  dilution  is  plated, 
and  we  find  180  colonies  on  the  plate.  Assuming  that 
each  colony  develops  from  an  individual  bacterium, 
though  this  is  perhaps  not  strictly  true,  we  had  180 
organisms  in  0.25  c.c.  of  our  1  :  100  dilution  ;  therefore 
in  0.25  c.c.  of  the  original  water  we  had  180  X  100  -= 
18,000  bacteria,  which  will  be  72,000  bacteria  per  cubic 
centimetre  (0.25  c.c.  =  18,000,  1  c.c.  =  18,000  X  4  = 
72,000).  The  results  are  always  to  be  expressed  in 
terms  of  the  number  of  bacteria  per  cubic  centimetre 
of  the  original  water. 

Another  point  of  very  great  importance  (already  men- 
tioned) is  the  effect  of  temperature  upon  the  number  of 
colonies  of  bacteria  that  will  develop  on  the  plates  made 
from  water.  It  must  always  be  remembered  that  a 
larger  number  of  colonies  appear  on  gelatin  plates  made 
from  water  and  kept  at  18°  to  20°  C.  than  on  agar-agar 
plates  kept  in  the  incubator.  The  following  table,  illus- 
trative of  this  point,  gives  the  results  of  parallel  anal- 
yses of  the  same  waters,  the  one  series  of  counts  having 
been  made  upon  gelatin  plates  at  the  ordinary  tempera- 
ture of  the  room,  the  other  upon  plates  of  agar-agar 


BACTERIOLOGICAL  STUDY  OF  WATER.        545 

kept  for  the  same  length  of  time  in  the  incubator  at 
from  37°  to  38°  C.  It  will  be  seen  from  the  table 
that  much  the  larger  number  of  colonies — i.  e.,  much 
higher  results — were  always  obtained  when  gelatin  was 
employed.  The  importance  of  this  point  in  the  quan- 
titative bacteriological  analysis  of  water  is  too  apparent 
to  require  further  comment. 

TABLE  COMPARING  THE  RESULTS  OBTAINED  BY  THE  USE  OF  GEL- 
ATIN AT  18P-200  C.  AND  AGAR-AGAR  AT  37°-38°  C.  IN  QUANTI- 
TATIVE BACTERIOLOGICAL  ANALYSES  OF  WATER.  RESULTS 
RECORDED  ARE  THE  NUMBER  OF  COLONIES  THAT  DEVELOPED 
FROM  THE  SAME  AMOUNT  OF  VARIOUS  WATERS  IN  EACH 
SERIES.i 

NUMBER  OF  COLONIES  FROM  WATER  THAT  DEVELOPED  UPON— 
Gelatin  plates  at  18°  to  20°  C.  Agar-agar  plates  at  37°  to  38°  C. 

310 170 

280 .   .      140 

310) J180 

340  J (160 

650) J210 

630J (320 

380) J290 

400 1 -  1210 

1000 ) f  100 

890  J (130 

340) J280 

370 J (210 

490 ) ......    f  110 

580  J (100 

Another  point  of  equal  importance  in  its  influence  upon 
the  number  of  colonies  that  develop  is  the  reaction  of  the 
gelatin.  A  marked  excess  of  either  alkalinity  or  acidity 
always  has  a  retarding  effect  upon  many  species  found  in 
water.  Fuller's  experience  at  the  Lawrence  (Mass.)  Ex- 

1  I  am  indebted  to  James  Homer  Wright,  Thomas  Scott  Fellow  in 
Hygiene  (1892-'93),  University  of  Pennsylvania,  for  the  results  pre- 
sented in  this  table. 
35 


546  BACTERIOLOGY. 

periment  Station  has  shown  that  gelatin  of  such  a  degree 
of  acidity  as  to  require  the  further  addition  of  from  15  to 
20  c.c.  per  litre  of  a  normal  caustic  alkali  solution  to  bring 
it  to  the  phenolphtalein  neutral  point  gives,  on  the  whole, 
the  best  results.  Thus,  by  way  of  illustration,  Fuller 
found  that  a  sample  of  Merrimac  River  water  gave 
5800  colonies  per  c.c.  on  phenolphtalein  neutral  gel- 
atin, 15,000  colonies  on  gelatin  that  would  need  20  c.c. 
of  normal  alkali  solution  to  bring  it  up  to  the  phenol- 
phtalein neutral  point — i.  e.,  a  feebly  acid  nutrient  gel- 
atin, and  500  colonies  on  a  gelatin  so  alkaline  as  to 
require  20  c.c.  of  a  normal  acid  solution  to  bring  it 
back  to  the  phenolphtalein  neutral  point. 

Throughout  this  part  of  the  work  it  is  to  be  borne  in 
mind  that  when  reference  is  made  to  plates  it  is  not  to 
a  set,  as  in  isolation  experiments,  but  to  a  single  plate. 

METHOD  OF  COUNTING  THE  COLONIES  ON  PLATES. 
— For  convenience  in  counting  colonies  on  plates  or  in 
tubes  it  is  customary  to  divide  the  whole  area  of  the 
gelatin  occupied  by  colonies  into  smaller  areas,  and 
either  count  all  the  colonies  in  each  of  these  areas  and 
add  the  several  sums  together  for  the  total,  or  to  count 
the  number  of  colonies  in  each  of  several  areas,  ten 
or  twelve,  take  the  mean  of  the  results  and  multiply 
this  by  the  number  of  areas  containing  colonies.  The 
latter  procedure  obtains,  of  course,  only  when  all  the 
areas  are  of  the  same  size.  By  this  method,  however, 
the  results  vary  so  much  in  different  counts  of  the  same 
plate  that  they  cannot  be  considered  as  more  than  rough 
approximations. 

NOTE. — Prepare  a  plate ;  calculate  the  number  of 
colonies  upon  it  by  this  latter  method.  Now  repeat 


BACTERIOLOGICAL  STUDY  OF  WATER.        547 

the  calculation,  making  the  average  from  another  set 
of  squares.  Now  actually  count  the  entire  number  of 
colonies  on  the  plate.  Compare  the  results. 

For  facilitating  the  counting  of  colonies  several  very 
convenient  devices  exist. 

WOLFFHUGEL'S  COUNTING-APPARATUS. — This  appa- 
ratus (Fig.  102)  consists  of  a  flat  wooden  stand,  the 
centre  of  which  is  cut  out  in  such  a  way  that  either  a 
black  or  white  glass  plate  may  be  placed  in  it.  These 
form  a  background  upon  which  the  colonies  may  more 
easily  be  seen  when  the  plate  to  be  counted  is  placed 

FIG.  102. 


Wolffhiigel's  apparatus  for  counting  colonies. 

upon  it.  When  the  gelatin  plate  containing  the  colonies 
has  been  placed  upon  this  background  of  glass  it  is 
covered  by  a  transparent  glass  plate  which  swings  on  a 
hinge.  This  plate,  which  is  ruled  in  square  centimetres 
and  subdivisions,  when  in  position  is  just  above  the 
colonies,  without  touching  them.  The  gelatin  plate  is 
moved  about  until  it  rests  under  the  centre  of  the  area 
occupied  by  the  ruled  lines.  The  number  of  colonies 


548  BACTERIOLOGY. 

in  each  square  centimetre  is  then  counted,  and  the  sum- 
total  of  the  colonies  in  all  these  areas  gives  the  number 
of  colonies  on  the  plate ;  or,  as  has  already  been  indi- 
cated, if  the  number  of  colonies  be  very  great,  a  mean 
may  be  taken  of  the  number  in  several  (six  or  eight) 
squares ;  this  is  to  be  multiplied  by  the  total  number 
of  squares  occupied  by  the  gelatin.  The  result  is  an 
approximation  of  the  total  number  of  colonies. 

When  the  colonies  are  quite  small,  as  is  frequently 
the  case,  the  counting  may  be  rendered  easier  by  the 
use  of  a  small  hand-lens.  (Fig.  103.) 

FIG.  103. 


Lens  for  counting  colonies. 

Several  useful  modifications  of  this  apparatus  have 
been  introduced.  The  most  important  is  that  of  Lafar.1 
Lafar's  counter  consists  of  a  glass  disk  of  the  diameter 
of  ordinary  size  Petri  dishes.  It  is  supplied  with  a  collar 
or  flange  that  fits  around  the  bottom  of  the  Petri  dish,  and 
thus  holds  the  counter  in  position.  The  disk  is  ruled  with 
concentric  circles,  and  its  area  is  divided  into  sectors  of 
such  sizes  that  the  spaces  between  the  concentric  circles 
and  the  radii  forming  the  sectors  are  of  equal  size. 
Three  of  the  sectors  are  subdivided  into  smaller  areas 
of  equal  size  for  convenience  in  counting  when  the 
colonies  are  very  numerous.  The  principles  involved 

1  Centralblatt  fiir  Bakteriologie  und  Parasitenkunde,  1891,  Ed.  xv. 
S.  331. 


BACTERIOLOGICAL  STUDY  OF  WATER        549 

are  similar  to  those  of  the  preceding  apparatus,  but  the 
circular  form  of  the  apparatus  admits  of  more  exactness 
when  counting  colonies  on  a  circular  plate.1 

Pakes 2  has  introduced  a  cheap  and  convenient  modi- 
fication of  Lafar^s  apparatus.  It  consists  of  a  sheet  of 
white  paper  on  which  is  printed  a  black  disk  ruled 


FIG.  104. 
16 


Of      ^ 

6  8 

Pakes's  apparatus  for  counting  colonies  (reduced  one-third). 

with  white  lines,  in  somewhat  the  same  fashion  as  is 
Lafar's  counter,  though  the  areas  of  the  smallest  sub- 
divisions are  not  of  one  size  and  do  not  bear  a  constant 

1  Lafar's  apparatus  is  to  be  obtained  from  F.  Mollenkopf,  10  Thor- 
strasse,  Stuttgart,  who  holds  the  patent  for  it.  Its  price  is  about  8 
marks. 

8  Journal  of  Bacteriology  and  Pathology,  1896,  vol.  iv.  No.  1. 


550  BACTERIOLOGY. 

relation  to  each  other.1  To  use  this  apparatus  (Fig. 
104)  the  Petri  dish  is  placed  centrally  upon  it,  the 
cover  of  the  dish  is  removed,  and  the  colonies  are 
counted  as  they  lie  over  the  spaces  bounded  by  the 
white  lines  on  the  black  disk  beneath.  When  the 
plate  is  centred  over  the  black  disk  the  portion  lying 
over  one  sector  is  exactly  one-sixteenth  of  the  whole 
plate. 

ESMARCH'S  COUNTER. — Esmarch  devised  a  counter 
(Fig.  105)  for  estimating  the  number  of  colonies  present 

FIG.  105. 


Esmarch's  apparatus  for  counting  colonies  in  rolled  tubes. 

upon  a  cylindrical  surface,  as  when  in  rolled  tubes. 
The  principles  and  methods  of  estimation  are  practically 
the  same  as  those  given  for  WolffhiigePs  apparatus. 
A  simpler  method  than   by  the  use  of  Esmarch's 

1  Copies  of  this  apparatus  are  to  be  had  of  Ash  &  Co.,  42  Southwark 
Street,  London,  or  of  Lentz  &  Sons,  North  Eleventh  Street,  Phila- 
delphia, Pa.  (The  cost  is  but  a  few  cents  per  copy.) 


BACTERIOLOGICAL  ANALYSIS  OF  AIR.        551 

apparatus  may  be  employed  for  counting  the  colonies 
in  rolled  tubes.  It  consists  in  dividing  the  tube  by 
lines  into  four  or  six  longitudinal  areas,  which  are  sub- 
divided by  transverse  lines  about  1  or  2  cm.  apart.  The 
lines  may  be  drawn  with  pen  and  ink.  They  need  not 
be  exactly  the  same  distance  apart  nor  exactly  straight. 
Beginning  with  one  of  these  squares  at  one  end  of  the 
tube,  which  may  be  marked  with  a  cross,  the  tube  is 
twisted  with  the  fingers,  always  in  one  direction,  and 
the  exact  number  of  colonies  in  each  square  as  it 
appears  in  rotation  is  counted,  care  being  taken  not  to 
count  a  square  more  than  once  ;  the  sums  are  then  added 
together,  and  the  result  gives  the  number  of  colonies  in 
the  tube.  This  method  may  be  facilitated  by  the  use 
of  a  hand-lens. 

In  all  these  methods  there  is  one  error  difficult  to 
eliminate  :  it  is  assumed  that  each  colony  has  grown 
from  a  single  organism.  This  is  probably  not  always 
the  case,  as  there  may  exist  clumps  of  bacteria  which 
represent  hundreds  or  even  thousands  of  individuals, 
but  which  still  give  rise  to  but  a  single  colony — ob- 
viously this  is  of  necessity  estimated  as  a  single  organ- 
ism in  the  water  under  analysis. 

Where  grounds  exist  for  suspecting  the  presence  of 
these  clumps  they  may  in  part  be  broken  up  by  shaking 
the  original  water  with  sterilized  sand. 

What  has  been  said  for  the  bacteriological  examina- 
tion of  water  holds  good  for  all  fluids  which  are  to  be 
subjected  to  this  form  of  analysis. 

BACTERIOLOGICAL   ANALYSIS   OF   AIR. 

Quite  a  number  of  methods  for  the  bacteriological 
study  of  the  air  exist.  In  the  main  they  consist  either 


552  BACTERIOLOGY. 

of  allowing  air  to  pass  over  solid  nutrient  media  (Koch, 
Hesse)  and  observing  the  colonies  which  develop  upon  the 
media,  or  of  filtering  the  bacteria  from  the  air  by  means 
of  porous  and  liquid  substances,  and  studying  the  organ- 
isms thus  obtained.  (Miguel,  Petri,  Strauss,  Wiirz, 
Sedgwick-Tucker.)  Because  of  their  greater  exactness, 
the  latter  have  supplanted  the  former  methods. 

In  some  of  the  methods  which  provide  for  the  filtra- 
tion of  bacteria  from  the  air  by  means  of  liquid  sub- 
stances a  measured  volume  of  air  is  aspirated  through 
liquefied  gelatin ;  this  is  then  rolled  into  an  Esmarch 
tube  and  the  number  of  colonies  counted,  just  as  was 
done  in  water  analysis.  This  is  the  simplest  procedure. 
An  objection  sometimes  raised  against  it  is  that  organisms 

FIG.  106. 


Petri's  apparatus  for  bacteriological  analysis  of  air.    The  tube 
packed  with  sand  is  seen  at  the  point  a. 

may  be  lost,  and  not  come  into  the  calculation,  by  pass- 
ing through  the  medium  in  the  centre  of  an  air-bubble 
without  being  arrested  by  the  fluid — an  objection  that 
appears  to  have  more  of  speculative  than  of  real  value. 


BACTERIOLOGICAL  ANALYSIS  OF  AIR.        553 

Filtration  through  porous  substances  appears,  on  the 
whole,  to  give  the  best  results.  Petri  recommends  as- 
piration of  a  measured  volume  of  air  through  glass 
tubes  into  which  sterilized  sand  is  packed.  (Fig.  106.) 
When  aspiration  is  finished  the  sand  is  mixed  with 
liquefied  gelatin,  plates  are  made,  and  the  number  of 
developing  colonies  counted,  the  results  giving  the 
number  of  organisms  contained  in  the  volume  of  air 
aspirated  through  the  sand. 

The  main  objection  to  this  method  is  the  possibility 
of  mistaking  a  sand-granule  for  a  colony.  This  objec- 
tion has  been  overcome  by  Sedgwick  and  Tucker,  who 
employ  granulated  sugar  instead  of  sand ;  this,  when 
brought  into  the  liquefied  gelatin,  dissolves,  and  no  such 
error  as  that  possible  in  the  Petri  method  can  be  made. 

SEDGWICK-TUCKER  METHOD. — On  the  whole,  the 
method  proposed  by  Sedgwick  and  Tucker  gives  such 
uniform  results  that  it  is  to  be  preferred  to  others.  It 
is  as  follows  : 

The  apparatus  employed  by  them  consists  essentially 
of  three  parts : 

1.  A  glass  tube  of  special  form,  to  which  the  name 
aerobioscope  has  been  given. 

2.  A  stout  copper  cylinder  of  about  sixteen  litres 
capacity,  provided  with  a  vacuum-gauge. 

3.  An  air-pump. 

The  aerobioscope  (Fig.  107)  is  about  35  cm.  in  its 
entire  length ;  it  is  15  cm.  long  and  4.5  cm.  in  diam- 
eter at  its  expanded  part ;  one  end  of  the  expanded  part 
is  narrowed  to  a  neck  2.5  cm.  in  diameter  and  2.5  cm. 
long.  To  the  other  end  is  fused  a  glass  tube  15  cm. 
long  and  0.5  cm.  inside  diameter,  in  which  is  to  be 
placed  the  filtering-material. 


554  BACTERIOLOGY. 

Upon  this  narrow  tube,  5  cm.  from  the  lower  end,  a 
mark  is  made  with  a  file,  and  up  to  this  mark  a  small 
roll  of  brass-wire  gauze  (a)  is  inserted ;  this  serves  as 
a  stop  for  the  filtering-material  which  is  to  be  placed 
over  it.  Beneath  the  gauze  (at  6),  and  also  at  the 
large  end  (c),  the  apparatus  is  plugged  with  cotton. 

FIG.  107. 


The  Sedgwick-Tucker  aerobioscope. 

When  thoroughly  cleaned,  dried,  and  plugged,  the 
apparatus  is  to  be  sterilized  in  the  hot-air  sterilizer. 
When  cool  the  cotton  plug  is  removed  from  the  large 
end  (c),  and  thoroughly  dried  and  sterilized  No.  50 
granulated  sugar  is  poured  in  until  it  just  fills  the 
10  cm.  (c?)  of  the  narrow  tube  above  the  wire  gauze. 
This  column  of  sugar  is  the  filtering-material  em- 
ployed to  engage  and  retain  the  bacteria.  After 
pouring  in  the  sugar  the  cotton-wool  plug  is  replaced, 
and  the  tube  is  again  sterilized  at  120°  C.  for  several 
hours. 

Taking  the  air  sample.  In  order  to  measure  the 
amount  of  air  used  the  value  of  each  degree  on  the 
vacuum-gauge  is  determined  in  terms  of  air  by  means 
of  an  air-meter,  or  by  calculation  from  the  known  ca- 
pacity of  the  cylinder.  This  fact  ascertained,  the  nega- 
tive pressure  indicated  by  the  needle  on  exhausting  the 
cylinder  shows  the  volume  of  air  which  must  pass  into 
it  in  order  to  fill  the  vacuum.  By  means  of  the  air- 


BACTERIOLOGICAL  ANALYSIS  OF  AIR.        555 

pump  one  exhausts  the  cylinder  until  the  needle 
reaches  the  mark  corresponding  to  the  amount  of  air 
required.1 

A  sterilized  aerobioscope  is  now  to  be  fixed  in  the 
upright  position  and  its  small  end  connected  by  a  rubber 
tube  with  a  stopcock  on  the  cylinder,  or  to  a  glass  tube 
tightly  fixed  in  the  neck  of  an  aspirating-bottle  by 
means  of  a  perforated  rubber  stopper.  The  cotton  plug 
is  then  moved  from  the  upper  end  of  the  aerobioscope, 
and  the  desired  amount  of  air  is  aspirated  through  the 
sugar.  Dust-particles  and  bacteria  will  be  held  back 
by  the  sugar.  During  manipulation  the  cotton  plug  is 
to  be  protected  from  contamination. 

When  the  required  amount  of  air  has  been  aspirated 
through  the  sugar  the  cotton  plug  is  replaced,  and  by 
gently  tapping  the  aerobioscope  while  held  in  an  almost 
horizontal  position  the  sugar,  and  with  it  the  bacteria, 
are  brought  into  the  large  part  (e)  of  the  apparatus. 
When  all  the  sugar  is  thus  shaken  down  into  this  part 
of  the  apparatus  about  20  c.c.  of  liquefied,  sterilized 
gelatin  is  poured  in  through  the  opening  at  the  end  c, 
the  sugar  dissolves,  and  the  whole  is  then  rolled  on  ice, 
just  as  is  done  in  the  preparation  of  an  ordinary  Esmarch 
tube. 

The  gelatin  is  most  easily  poured  into  the  aerobio- 
scope by  the  use  of  a  small,  sterilized,  cylindrical  funnel 
(Fig.  108),  the  stem  of  which  is  bent  to  an  angle  of 
about  110°  with  the  long  axis  of  the  body. 

1  Such  a  cylinder  and  air-pump  are  not  necessary.  A  pair  of  ordinary 
aspirating-bottles  of  known  capacity  graduated  into  litres  and  fractions 
thereof  answer  perfectly  well.  Or  one  can  determine  by  the  weight 
of  water  that  has  flowed  from  the  aspirator  the  volume  of  air  that  has 
passed  in  to  take  its  place— t.  e.,  the  volume  of  air  that  has  passed 
through  the  aerobioscope. 


556 


BACTERIOLOGY. 


The  larger  part  of  the  aerobioscope  is  divided  into 
squares  to  facilitate  the  counting  of  the  colonies. 

By  the  employment  of  this  apparatus  one  can  filter 
the  air  at  any  place,  and  can  then,  without  fear  of  con- 
tamination, carry  the  tubes  to  the  laboratory  and  com- 
plete the  analysis.  Aside  from  this  advantage,  the  filter 

FIG.  108. 


Bent  funnel  for  use  with  aerobioscope. 

being  soluble  only  the  insoluble  bacteria  are  left  im- 
bedded in  the  gelatin. 

For  general  use  this  method  is  to  be  preferred  to  the 
others  that  have  been  mentioned. 

BACTERIOLOGICAL    STUDY   OF   THE   SOIL. 

Bacteriological  study  of  the  soil  may  be  made  by 
either  breaking  up  small  particles  of  earth  in  liquefied 
media  and  making  plates  directly  from  this ;  or  by  what 
is  perhaps  a  better  method,  as  it  gets  rid  of  insoluble 
particles  which  may  give  rise  to  errors :  breaking  up 


BACTERIOLOGICAL  STUDY  OF  THE  SOIL.     557 

the   soil  in  sterilized  water  and  then   making   platen 
immediately  from  the  water. 

It  must  be  borne  in  mind  that  many  of  the  ground- 
organisms  belong  to  the  anaerobic  group,  so  that  in 
these  studies  this  point  should  be  remembered  and  the 
methods  for  the  cultivation  of  such  organisms  practised 
in  connection  with  the  ordinary  methods.  It  must  also 
be  remembered  that  the  nitrifying  organisms,  every- 
where present  in  the  ground,  cannot  be  isolated  by  the 
ordinary  methods,  and  will  not  appear  in  plates  made 
after  either  of  the  above  plans.  The  special  devices 
for  their  cultivation  are  described  in  the  chapter  on 
soil-organisms. 


CHAPTER  XXVIII. 

Methods  of  testing  disinfectants  and  antiseptics — Experiments  illus- 
trating the  precautions  to  be  taken — Experiments  in  skin-disin- 
fection. 

DETERMINATION   OF   DISINFECTANT   PROPERTIES. 

THERE  are  several  ways  of  determining  the  germicidal 
value  of  chemical  substances,  the  most  common  being 
to  expose  organisms  dried  upon  bits  of  silk  thread  to 
the  disinfectant  for  different  lengths  of  time,  and  then, 
after  removing,  and  carefully  washing  the  threads  in 
water,  to  place  them  in  nutrient  media  at  a  favorable 
temperature,  and  notice  if  any  growth  appears.  If  no 
growth  results,  the  disinfection  is  presumably  successful. 
Another  method  is  to  mix  fluid  cultures  of  bacteria  with 
the  disinfectant  in  varying  proportions,  and,  after  dif- 
ferent intervals  of  time,  to  determine  if  disinfection  is 
in  progress  by  transferring  a  portion  of  the  mixture  to 
nutrient  media,  just  as  in  the  other  methods  of  work. 

By  the  first  of  these  processes  the  bits  of  thread, 
usually  about  1  to  2  cm.  long,  are  placed  in  a  dry  test- 
tube  provided  with  a  cotton  plug  and  carefully  sterilized, 
either  by  the  dry  method  or  in  the  steam  sterilizer, 
before  using.  They  are  then  immersed  in  a  pure 
bouillon  culture  or  in  a  salt-solution  suspension  of  the 
organism  upon  which  the  disinfectant  is  to  be  tested. 
I  say  "pure  culture/7  because  it  is  always  desirable  in 
testing  a  substance  to  determine  its  germicidal  value 

558 


METHODS  OF  TESTING  DISINFECTANTS.      559 

for  several  different  resistant  species  of  bacteria,  both 
in  the  vegetating  and  in  the  spore  stage,  and  also  be- 
cause it  is  only  by  the  use  of  pure  cultures  of  familiar 
species  that  it  is  possible  to  distinguish  between  the 
colonies  growing  from  the  individuals  that  have  not 
been  destroyed  by  the  disinfectant  under  investigation 
and  those  of  unknown  species  that  may  appear  upon 
the  plate  as  contaminations  occurring  during  the  manip- 
ulation. 

After  the  threads  have  remained  in  the  culture  or 
suspension  for  from  five  to  ten  minutes  they  are  re- 
moved under  antiseptic  precautions  and  carefully  sepa- 
rated and  spread  upon  the  bottom  of  a  sterilized  Petri 
dish,  which  is  then  placed  either  in  the  incubator  at  a 
temperature  not  exceeding  38°  C.  until  the  excess  of  fluid 
has  evaporated,  or  in  a  desiccator  over  sulphuric  acid, 
calcium  chloride,  or  any  other  drying-agent.  The  threads 
are  not  left  there  until  absolutely  dry,  but  only  until  the 
excess  of  moisture  has  evaporated.  When  sufficiently  dry 
they  are  immersed  in  solutions  of  the  disinfectant  of  dif- 
ferent but  known  strengths  for  a  fixed  interval  of  time, 
say  one  or  two  hours,  after  which  they  are  removed, 
rinsed  in  sterilized  distilled  water  to  remove  the  ex- 
cess of  disinfectant  adhering  to  them,  and  placed  in 
fresh,  sterile  culture-media,  which  are  then  placed  in 
the  incubator  at  from  37°  to  38°  C.  If  after  twenty- 
four  to  forty-eight  or  seventy-two  hours  a  growth  occurs 
at  or  about  the  bit  of  thread,  and  if  this  growth  consists 
of  the  organism  with  which  the  test  was  made,  mani- 
festly there  has  been  no  disinfection ;  if  no  growth 
occurs  after,  at  most,  ninety-six  hours,  it  is  safe  to  pre- 
sume that  the  bacteria  have  been  killed,  unless  our 
efforts  at  rinsing  off  the  excess  of  disinfectant  from 


560  BACTERIOLOGY. 

the  thread  have  not  been  successful,  and  a  small  amount 
of  disinfectant  is  still  active  in  preventing  development 
— i.  e.j  is  acting  as  an  antiseptic. 

By  the  process  in  which  cultures  or  suspensions 
of  the  organisms  are  mixed  with  different  but  known 
strengths  of  the  disinfectant  a  small  portion  of  the 
mixture,  usually  a  loopful  or  a  drop,  is  transferred 
at  the  end  of  a  definite  time  to  the  fresh  medium 
which  is  to  determine  whether  the  organisms  have 
been  killed  or  not.  This  is  commonly  a  tube  of  fluid 
agar-agar,  which  is  poured  into  a  Petri  dish,  allowed  to 
solidify,  and  placed  in  the  incubator,  as  in  the  preceding 
method. 

After  the  minimum  strength  of  disinfectant  necessary 
to  destroy  the  vitality  of  the  organisms  with  which  we 
are  working  has  been  determined  for  any  fixed  time,  it 
remains  for  us  to  decide  what  is  the  shortest  time  in  which 
this  strength  will  have  the  same  effect.  We  then  work 
with  a  constant  dilution  of  the  disinfectant,  but  with 
varying  intervals  of  exposure — one,  five,  ten  minutes, 
etc. — until  we  have  decided  not  only  the  minimum 
amount  of  Disinfectant  required  for  the  destruction  of 
the  bacteria,  but  the  shortest  time  necessary  for  this 
under  known  conditions. 

A  factor  not  to  be  lost  sight  of  is  the  temperature 
at  which  these  experiments  are  conducted,  for  it 
must  always  be  borne  in  mind  that  the  action  of  a  dis- 
infectant is  usually  more  energetic  at  a  higher  than  at  a 
lower  temperature. 

Now  in  both  of  these  methods  it  is  easy  to  see  that 
unless  special  precautions  are  taken  a  minute  portion  of 
the  disinfectant  may  be  carried  along  with  the  thread, 
or  drop,  into  the  medium  which  is  to  determine  whether 


METHODS  OF  TESTING  DISINFECTANTS,      561 

the  organisms  do  or  do  Dot  possess  the  power  of  growth, 
and  there  have  a  restraining  or  antiseptic  action.  For 
organisms  in  their  normal  condition — that  is,  those 
which  have  never  been  exposed  to  the  action  of  a  dis- 
infectant— the  amount  of  certain  disinfectants  that  is 
i  yy  :sary  to  restrain  growth  is  very  small  indeed  ;  and 
t»-  organisms  that  have  already  been  exposed  for  a 
ime  to  such  agents  this  amount  is  very  much  less. 
It  is  plain,  then,  that  if  the  test  is  to  be  an  accurate 
one,  precautions  must  be  taken  against  admitting  this 
minute  trace  of  disinfectant  to  the  medium  with  which 
we  are  to  determine  whether  the  bacteria  exposed  to  the 
disinfectant  were  killed  or  not.  The  precautions  hitherto 
taken  to  prevent  this  accident  have  been,  when  the  threads 
were  employed,  washing  them  in  sterilized  distilled 
water  and  then  in  alcohol ;  or,  where  fluid  cultures  were 
mixed  with  the  disinfectant  in  solution,  an  effort  was 
usually  made  to  dilute  the  amount  of  disinfectant  car- 
ried over,  to  a  point  at  which  it  lost  its  inhibiting 
power. 

While  such  precautions  are  sufficient  in  many  cases, 
they  do  not  answer  for  all.  Certain  chemicals  have  the 
property  of  combining  so  firmly  with  the  threads  upon 
which  the  bacteria  are  located  as  to  require  other  special 
means  of  ridding  the  threads  of  them  ;  and  in  solutions 
in  which  proteid  substances  are  present  along  with  the 
bacteria  a  similar  union  between  them  and  the  disin- 
fectant may  likewise  take  place.  In  both  instances  this 
amount  of  disinfectant  adhering  to  the  threads  or  in 
combination  with  the  proteids  must  be  gotten  rid  of, 
otherwise  the  results  of  the  test  may  be  fallacious.  A  par- 
tial solution  of  the  problem  is  given  through  studies  that 
have  been  made  upon  corrosive  sublimate  in  its  various 


562  BACTERIOLOGY. 

applications  for  disinfecting  purposes,  and  in  this  con- 
nection it  has  been  shown  by  Shaefer l  that  it  is  impos- 
sible to  rid  silk  threads  of  the  corrosive  sublimate  ad- 
hering to  them  by  simple  washing,  as  the  sublimate 
acts  as  a  mordant  and  forms  a  firm  union  with  the  tis- 
sues of  the  threads.  Braatz  2  found  the  same  to  hold 
good  for  catgut.  For  example,  he  found  that  catgut  which 
had  been  immersed  in  solutions  of  corrosive  sublimate 
gave  the  characteristic  reactions  of  the  salt  after  having 
been  immersed  for  five  weeks  in  distilled  water  which 
had  been  repeatedly  renewed.  Braatz  remarks  that 
a  similar  combination  between  sublimate  and  cotton 
will  take  place  after  a  long  time  ;  but  it  occurs  so 
slowly  that  it  cannot  interfere  with  disinfection-experi- 
ments in  the  same  way  that  silk  does. 

The  most  successful  attempt  at  removing  all  traces 
of  sublimate  from  the  threads  or  from  the  proteid  sub- 
stances in  which  are  located  the  bacteria  whose  vitality 
is  to  be  tested  wras  made  by  Geppert,  who  subjected 
them  to  the  action  of  ammonium  sulphide  in  solution. 
By  this  procedure  the  mercury  is  converted  into  inert, 
insoluble  sulphide,  and  has  no  inhibiting  effect  upon  the 
growth  of  those  bacteria  that  did  not  succumb  to.  its 
action  when  in  the  form  of  the  bichloride. 

In  the  second  method  of  testing  disinfectants  men- 
tioned above — that  is,  when  cultures  of  bacteria  and 
solutions  of  the  disinfectant  are  mixed,  and  after  a  time 
a  drop  of  the  mixture  is  removed  and  added  to  sterile 
nutrient  media — the  inhibiting  amount  of  disinfectant 
can  readily  be  gotten  rid  of  by  dilution  ;  that  is  to  say, 

1  Shaefer  :  Berliner  klin.  Wochenschrift,  1890,  No.  3,  p.  50. 

2  Braatz  :  Centralblatt  fur  Bakteriolgie  und  Parasiteukunde,  Bd.  vii. 
No.  1,  p.  8. 


METHODS  OF  TESTING  DISINFECTANTS.      563 

instead  of  transferring  the  drop  directly  to  the  fresh 
medium,  add  it  to  10  or  12  c.c.  of  sterilized  salt-solu- 
tion (0.6-0.7  per  cent,  of  NaCl  in  distilled  water)  or 
distilled  water,  and  after  thoroughly  shaking  add  a  drop 
of  this  to  the  medium  in  which  the  power  of  develop- 
ment of  the  bacteria  is  to  be  determined. 

Another  important  point  to  be  borne  in  mind  in 
testing  disinfectants  is  the  necessity  of  so  adjusting  the 
conditions  that  each  individual  organism  will  be  ex- 
posed to  the  action  of  the  agent  used.  When  clumps 
of  bacteria  exist  we  are  not  always  assured  of  this,  for 
only  those  on  the  surface  of  the  clump  may  be  affected, 
while  those  in  the  centre  of  the  mass  may  escape,  being 
protected  by  those  surrounding  them.  These  clumps 
and  minute  masses  are  especially  liable  to  be  present 
in  fluid  cultures  and  in  suspensions  of  bacteria,  and 
must  be  eliminated  before  the  test  is  begun,  if  this  is  to 
be  made  by  mixing  them  with  solutions  of  the  agent  to 
be  tested.  This  is  best  accomplished  in  the  following 
way :  the  organisms  should  be  cultivated  in  bouillon 
containing  sand  or  finely  divided  particles  of  glass; 
after  growing  for  a  sufficient  length  of  time  they  are 
to  be  shaken  thoroughly,  in  order  that  all  clumps  may 
be  mechanically  broken  up  by  the  sand.  The  culture  is 
then  filtered  through  a  tube  containing  closely  packed 
glass-wool. 

The  filtration  may  be  accomplished  without  fear  of 
contamination  of  the  culture  by  the  employment  of  an 
Allihin  tube,  which  is  practically  a  thick-walled  test- 
tube  drawn  out  to  a  finer  tube  at  its  blunt  end  so  as  to 
convert  it  into  a  sort  of  cylindrical  funnel.  The  tube 
when  ready  for  use  has  the  appearance  shown  in  Fig. 
109. 


564 


BACTERIOLOGY. 


FIG.  109. 


This  tube,  after  being  plugged  at  the  bottom  with 
glass-wool  (a,  Fig.  109),  and  at  its  wide  extremity 
with  cotton-wool,  is  placed  vertically, 
small  end  down,  into  an  Erlenmeyer 
flask  of  about  100  c.c.  capacity  and 
sterilized  in  a  steam  sterilizer  for  the 
proper  time.  It  is  kept  in  the  sterilizer 
until  it  is  to  be  used,  which  should  be 
as  soon  as  possible  after  sterilization. 

The  watery  suspension  or  bouillon 
culture  of  the  organisms  is  now  to  be 
filtered  repeatedly  through  the  glass- 
wool  into  sterilized  flasks  until  a  de- 
gree of  transparency  is  reached  which 
will  permit  the  reading  of  moderately 
fine  print  through  a  layer  of  the  fluid 
about  2  cm.  thick — i.  e.9  through  an  or- 
dinary test-tube  full  of  it.  This  filtrate 
can  then  be  subjected  to  the  action  of 
the  disinfectant.  As  a  rule,  the  results 
are  more  uniform  than  when  no  atten- 
tion is  paid  to  the  presence  of  clumps. 
It  is  scarcely  necessary  to  say  that  in 
the  practical  employment  of  disinfec- 
tants outside  the  laboratory  no  such  pre- 
cautions are  taken ;  but  in  laboratory 
work,  where  it  is  desired  to  determine 
exactly  the  value  of  different  substances 
as  germicides,  all  the  precautions  men- 
tioned will  be  found  essential  to  preci- 
sion. 

The  disinfectant  value  of  gases  and  vapors  is  deter- 
mined   by   their    action    upon    test-objects   in    closed 


Cylindrical  fun- 
nel used  for  filtering 
cultures  on  which 
disinfectants  are  to 
be  tested. 


METHODS  OF  TESTING  DISINFECTANTS.      565 

chambers.  The  object  is  to  determine  the  proportion 
of  the  gas,  when  mixed  with  air,  that  is  required  to 
destroy  the  bacteria  exposed  to  its  action  in  a  given 
time.  For  this  purpose  the  test  is  usually  made  as 
follows  :  under  a  sterilized  bell-glass  of  known  capacity 
the  test-objects  are  placed.  Into  the  chamber  is  then 
admitted  sufficient  of  a  mixture  of  air  and  the  gas  under 
consideration,  of  known  proportions,  to  displace  com- 
pletely all  the  air ;  or  the  pure  gas  itself  may  be  intro- 
duced in  amount  necessary  to  give  the  desired  dilution 
when  mixed  with  the  air  in  the  chamber.  At  the 
expiration  of  the  time  decided  upon  for  the  test  the 
infected  articles  are  removed  and  the  vitality  of  the 
bacteria  upon  them  is  determined. 

In  the  case  of  vapors  of  volatile  fluids,  such,  for 
instance,  as  formalin,  the  fluid  is  placed  under  the  bell- 
glass  in  an  open  dish ;  in  another  open  dish  the  test- 
objects  are  placed.  The  bell-glass  is  then  sealed  to  an 
underlying  ground-glass  plate  by  vaselin  or  paraffin, 
and  the  fluid  is  allowed  to  vaporize  at  ordinary  room- 
temperature.  The  point  here  to  be  decided  is  the  vol- 
ume or  weight  of  such  a  fluid  that  it  is  necessary  to 
expose  in  an  air-chamber  of  known  cubic  capacity  in 
order  that  bacteria  may  be  destroyed  by  its  vapor  in  a 
given  time. 

In  determining  the  germicidal  value  of  different 
chemical  agents  for  certain  pathogenic  bacteria  sus- 
ceptible animals  are  sometimes  inoculated  with  the 
organisms  after  they  have  been  exposed  to  the  disin- 
fectant. If  no  pathological  condition  results,  disinfec- 
tion is  presumed  to  have  been  successful ;  while  if  the 
condition  characteristic  of  the  activities  of  the  given 
organism  in  the  tissues  of  this  animal  appears,  the 


566  BACTERIOLOGY. 

reverse  is  the  case.  The  objections  to  this  method 
are :  "  First.  The  test-organisms  may  be  modified  as 
regards  reproductive  activity  without  being  killed ; 
and  in  this  case  a  modified  form  of  the  disease  may 
result  from  the  inoculation,  of  so  mild  a  character 
as  to  escape  observation.  Second.  An  animal  that 
has  suffered  this  modified  form  of  the  disease  enjoys 
protection,  more  or  less  perfect,  from  future  attacks, 
and  if  used  for  a  subsequent  experiment  may,  by 
its  immunity  from  the  effects  of  the  pathogenic  test- 
organism,  give  rise  to  the  mistaken  assumption  that 
this  had  been  destroyed  by  the  action  of  the  germicidal 
agent  to  which  it  had  been  subjected."  (Sternberg.) 

DETERMINATION    OF   ANTISEPTIC   PROPERTIES. 

For  this  purpose  sterile  media  are  employed,  and  are 
usually  arranged  in  two  groups :  the  one  to  remain 
normal  in  composition  and  to  serve  as  controls,  while  to 
the  other  the  substance  to  be  tested  is  to  be  added  in  dif- 
ferent but  known  strengths.  It  is  customary  to  employ 
test-tubes  each  containing  an  exact  amount  of  bouillon, 
gelatin,  or  agar-agar,  as  the  case  may  be.  To  each  tube 
a  definite  amount  of  the  antiseptic  is  added,  and  if  it 
is  not  of  a  volatile  nature  or  not  injured  by  heat,  the 
tubes  may  then  be  sterilized.  After  this  they  are  to  be 
inoculated  with  the  organism  with  which  the  test  is  to 
be  made,  and  at  the  same  time  one  of  the  "  control  "- 
tubes  (one  of  those  to  which  no  antiseptic  has  been 
added)  is  inoculated.  They  are  all  then  to  be  placed 
in  the  incubator  and  kept  under  observation.  If  at  the 
end  of  twenty-four,  forty-eight,  or  seventy-two  hours 
no  growth  appears  in  any  but  the  "  control  "-tubes,  it 
is  evident  that  the  antiseptic  must  be  added  in  smaller 


EXPERIMENTS.  567 

amounts,  for  we  are  to  determine  the  point  at  which  it 
/*  not  as  well  as  that  at  which  it  is  capable  of  preventing 
development.  The  experiment  is  then  repeated,  using 
smaller  amounts  of  the  antiseptic  until  we  reach  a  point 
at  which  growth  just  occurs,  notwithstanding  the  pres- 
ence of  the  antiseptic  ;  the  amount  necessary  for  antisep- 
sis is  then  a  trifle  greater  than  that  used  in  the  last  tube. 
If,  for  example,  there  was  no  development  in  the  tubes 
in  which  the  antiseptic  svas  present  in  the  proportion 
of  1  :  1000,  and  growth  in  the  one  in  which  it  was 
present  in  1  :  1400,  the  experiment  should  be  repeated 
with  strengths  of  the  antiseptic  corresponding  to 
1  : 1000,  1  : 1100,  1  :  1200,  1  : 1300,  1  : 1400,  and  in 
this  way  one  ultimately  determines  the  amount  by  which 
growth  is  just  prevented  ;  this  represents  the  antiseptic 
value  of  the  substance  for  the  organism  with  which  it 
was  tested. 

EXPERIMENTS. 

To  each  of  three  tubes  containing  10  c.c. — one  of 
physiological  salt-solution,  another  of  bouillon,  a  third  of 
fluid  blood-serum — add  as  much  of  a  culture  of  staphylo- 
cocciis  pyogenes  aureus  as  can  be  held  upon  a  looped 
platinum  wire.  Break  this  up  carefully  to  eliminate 
clumps,  and  then  add  exactly  10  c.c.  of  a  1  : 500  solution 
of  corrosive  sublimate.  Mix  thoroughly,  and  at  the 
end  of  three  minutes  transfer  a  drop  from  each  tube 
into  tubes  of  liquefied  agar-agar,  and  pour  these  into 
Petri  dishes.  Label  each  dish  carefully  and  place  them 
in  the  incubator.  Are  the  results  the  same  in  all  the 
plates  ?  How  are  the  differences  to  be  explained  ?  To 
what  strength  of  the  disinfectant  were  the  organisms 
exposed  in  the  experiment? 


568  BACTERIOLOGY. 

To  each  of  two  tubes — the  one  containing  10  c.c. 
of  normal  salt-solution,  the  other  of  bouillon — add  as 
much  of  a  spore-containing  culture  of  anthrax  bacilli 
as  can  be  held  upon  a  loop  of  platinum  wire.  Dis- 
tribute this  uniformly  through  the  medium,  and  then 
add  exactly  10  c.c.  of  a  1  :  500  solution  of  corrosive 
sublimate.  Mix  thoroughly,  and  at  the  end  of  five 
minutes  transfer  a  drop  from  each  tube  to  tubes  of 
liquefied  agar-agar.  Pour  these  immediately  into 
Petri  dishes.  Label  each  dish  carefully  and  place  them 
in  the  incubator.  Note  the  results  at  the  end  of  twenty- 
four,  forty-eight,  and  seventy-two  hours.  How  do 
you  explain  them  ? 

Make  identically  the  same  experiment  with  the  same 
spore-containing  culture  of  anthrax  bacilli,  except  that 
the  drop  from  the  mixture  is  to  be  transferred  to  10  c.c. 
of  a  mixture  of  equal  parts  of  ammonium  sulphide  and 
sterilized  distilled  water.  After  remaining  in  this  for 
about  half  a  minute,  a  drop  is  to  be  transferred  to  a 
tube  of  liquefied  agar-agar,  poured  into  Petri  dishes, 
labelled,  and  placed  in  the  incubator.  Note  the  results. 
Do  they  correspond  with  those  obtained  in  the  pre- 
ceding experiment?  How  are  the  differences  ex- 
plained ? 

Prepare  a  1  : 1000  solution  of  corrosive  sublimate. 
.To  each  of  twelve  tubes  containing  exactly  10  c.c.  of 
bouillon  add  one  drop  to  the  first,  two  drops  to  the 
second,  and  so  on  until  the  last  tube  has  had  twelve 
drops  added  to  it.  Mix  thoroughly  and  then  inoculate 
each  with  one  wire-loopful  of  a  bouillon  culture  of 
staphylococcus  pyogenes  aureus.  Place  them  all  in  the 


EXPERIMENTS.  569 

incubator  after  carefully  labelling  them.     Note  the  order 
in  which  growth  appears. 

Do  the  same  with  anthrax  spores,  with  spores  of 
bacillus  subtilis,  and  with  the  typhoid  bacillus,  and  com- 
pare the  results.  From  these  experiments,  what  will  be 
the  strength  of  corrosive  sublimate  necessary  to  anti- 
sepsis under  these  conditions  for  the  organisms  em- 
ployed ? 

Make  a  similar  series  of  experiments  using  a  5  per 
cent,  solution  of  carbolic  acid. 

Determine  the  antiseptic  value  of  the  common  disin- 
fectants for  the  organisms  with  which  you  are  working. 

Determine  the  time  necessary  for  the  destruction  of 
the  organisms  with  which  you  are  working,  by  corro- 
sive sublimate  in  1  : 1000  solution,  under  different  con- 
ditions— with  and  without  the  presence  of  albuminous 
bodies  other  than  the  bacteria,  and  under  varying  con- 
ditions of  temperature. 

In  making  these  experiments  be  careful  to  guard 
against  the  introduction  of  sufficient  sublimate  into  the 
agar-agar  with  which  the  Petri  plate  is  to  be  made  to 
inhibit  the  growth  of  the  organisms  which  may  not  have 
been  destroyed  by  the  sublimate.  This  may  be  done  by 
transferring  two  drops  from  the  mixture  of  sublimate 
and  organism  into  not  less  than  10  c.c.  of  sterilized 
physiological  salt-solution,  in  which  they  may  be  thor- 
oughly shaken  for  from  one  to  two  minutes,  or  into  the 
solution  of  ammonium  sulphide  of  the  strength  given. 

To  10  c.c.  of  a  bouillon  culture  of  §taphylococcu8 
pyogenes  aureus  or  anthrax  spores  add  10  c.c.  of  a 


570  BACTERIOLOGY. 

1  :  500  solution  of  corrosive  sublimate,  and  allow  it  to 
remain  in  contact  with  the  organisms  for  only  one-hcdf 
the  time  necessary  to  destroy  them  (use  an  organism 
for  which  this  has  been  determined).  Then  transfer  a 
drop  of  the  mixture  to  each  of  three  liquefied  agar-agar 
tubes  and  pour  them  into  Petri  dishes.  Place  them  in 
the  incubator  and  observe  them  for  twenty-four,  forty- 
eight,  and  seventy-two  hours.  No  growth  occurs.  How 
is  this  to  be  accounted  for  ? 

At  the  end  of  seventy-two  hours  inoculate  all  of  these 
plates  with  a  culture  of  the  same  organism  which  has 
not  been  exposed  to  sublimate,  by  taking  up  bits  of  cult- 
ure on  a  needle  and  drawing  it  across  the  plates.  A 
growth  now  results.  We  have  here  an  experiment  in 
which  organisms  which  have  been  exposed  to  sublimate 
for  a  much  shorter  time  than  necessary  to  destroy  them, 
when  transferred  directly  to  a  favorable  culture-medium 
do  not  grow,  and  yet,  when  the  same  organism  which 
has  not  been  exposed  to  sublimate  at  all  is  planted  upon 
the  same  medium  it  does  grow.  How  is  this  to  be  ac- 
counted for? 

SKIN-DISINFECTION. — With  a  sterilized  knife  scrape 
from  the  skin  of  the  hands,  at  the  root  of  the  nails,  and 
under  the  nails,  small  particles  of  epidermis.  Prepare 
plates  from  them.  Note  the  results. 

Wash  the  hands  carefully  for  ten  minutes  in  hot 
water  and  scrub  them  during  this  time  with  soap  and 
a  sterilized  brush.  Rinse  them  in  hot  water.  Again 
prepare  plates  from  scrapings  of  the  skin  on  the  fingers, 
at  the  root  of  the  nails,  and  under  the  nails.  Note  the 
results. 

Again  wash  as  before  in  hot  water  with   soap  and 


EXPERIMENTS.  571 

brush,  rinse  in  hot  water,  then  soak  the  hands  for  live 
minutes  in  1  : 1000  corrosive  sublimate  solution,  and, 
as  before,  prepare  plates  from  scrapings  from  the  same 
localities.  Note  the  results. 

Repeat  this  latter  procedure  in  exactly  the  same  way, 
but  before  taking  the  scrapings  let  some  one  pour  am- 
monium sulphide  over  the  points  from  which  the  scrap- 
ings are  to  be  made.  After  it  has  been  on  the  hands 
about  three  minutes  again  scrape,  and  note  the  result 
upon  plates  made  from  the  scrapings. 

Wash  as  before  in  hot  water  and  soap,  rinse  in  clean 
hot  water,  immerse  for  a  minute  or  two  in  alcohol, 
after  this  in  1  : 1000  sublimate  solution,  and  finally  in 
ammonium  sulphide,  and  then  prepare  plates  from 
scrapings  from  the  points  mentioned. 

In  what  way  do  the  results  of  these  experiments 
differ  from  one  another? 

To  what  are  these  differences  due  ? 

What  have  these  experiments  taught? 

In  making  the  above  experiments  it  must  be  remem- 
bered that  the  strictest  care  is  necessary  in  order  to 
prevent  the  access  of  germs  from  without  into  our 
media.  The  hand  upon  which  the  experiment  is  being 
performed  must  be  held  away  from  the  body  and  must 
not  touch  any  object  not  concerned  in  the  experiment. 
The  scraping  should  be  done  with  the  point  of  a  knife 
that  has  been  sterilized  in  a  flame  and  allowed  to  cool. 
The  scrapings  may  be  transferred  directly  from  the 
knife-point  to  the  gelatin  by  means  of  a  sterilized  plat- 
inum wire  loop. 

The  brush  used  should  be  thoroughly  cleansed  and 
always  kept  in  1  : 1000  solution  of  corrosive  sublimate. 
It  should  be  washed  in  hot  water  before  using. 


APPENDIX. 


LIST  of  apparatus  and  materials  required  in  a  begin- 
ner's bacteriological  laboratory : 

MICROSCOPE   AND   ACCESSORIES. 

Microscope  with  coarse  and  fine  adjustment  and 
heavy,  firm  base;  Abbe  sub-stage  condensing  system, 
arranged  either  as  the  "  simple  "  or  as  the  regular  Abbe 
condenser,  in  either  case  to  be  provided  with  iris  dia- 
phragm ;  objectives  equivalent,  in  the  English  nomen- 
clature, to  about  one-fourth  inch  and  one-sixth  inch 
dry,  and  one-twelfth  inch  oil-immersion  system;  a 
triple  revolving  nose-piece ;  three  oculars,  varying  in 
magnifying  power ;  and  a  bottle  of  immersion  oil. 

Glass  slides,  English  shape  and  size  and  of  colorless 
glass. 

Six  slides  with  depressions  of  about  1  cm.  in  diameter 
in  centre. 

Cover-slips,  15  by  15  mm.  square  and  not  more  than 
from  0.15  to  0.18  mm.  thick. 

Forceps.  One  pair  of  fine-pointed  forceps  and  one 
pair  of  the  Cornet  or  Stewart  pattern,  for  holding  cover- 
slips. 

Platinum  needles  with  glass  handles.  One  straight, 
about  4  cm.  long ;  one  looped  at  the  end,  about  4  cm. 
long;  and  one  straight,  about  8  cm.  long.  Glass 

573 


574  APPENDIX. 

handles  to  be  about  3  mm.  in  thickness  and  from  15 
to  17  cm.  long. 

STAINING-   AND   MOUNTING-REAGENTS. 

200  c.c.  of  saturated  alcoholic  solution  of  fuchsin. 

200  c.c.  of  saturated  alcoholic  solution  of  gentian- 
violet. 

200  c.c.  of  saturated  alcoholic  solution  of  methylene- 
blue. 

200  grammes  of  pure  aniline. 

200  grammes  of  C.  P.  carbolic  acid. 

500  grammes  of  C.  P.  nitric  acid. 

500  grammes  of  C.  P.  sulphuric  acid. 

200  grammes  of  C.  P.  glacial  acetic  acid. 

1  litre  of  ordinary  93-95  per  cent,  alcohol. 

1  litre  of  absolute  alcohol. 
500  grammes  of  ether. 

500  grammes  of  pure  xylol. 

50  grammes  of  Canada  balsam  dissolved  in  xylol. 
100  grammes  of  Schering's  celloidin. 
10  grammes  of  iodine  and  30  grammes  of  potassium 
iodide  in  substance. 

100  grammes  of  tannic  acid. 
100  grammes  of  ferrous  sulphate. 
Distilled  water. 

FOR   NUTRIENT   MEDIA. 

J  pound  of  Liebig's  or  Armour's  beef-extract. 
250  grammes  of  Witte's  or  Sargent's  peptone. 

2  kilogrammes  of  gold-label  gelatin  (Hesteberg's). 
1 00  grammes  of  agar-agar  in  substance. 

200  grammes  of  sodium  chloride  (ordinary  table-salt). 
500  grammes  of  pure  glycerin. 


APPENDIX.  575 

50  grammes  of  pure  glucose. 

20  grammes  of  pure  lactose. 

100  grammes  of  caustic  potash. 

200  c.c.  of  litmus  tincture. 

10  grammes  of  rosolic  acid  (corallin). 

Blue  and  red  litmus-paper ;  curcuma  paper. 

5  grammes  of  phenolphtalein  in  substance. 
Filter-paper,  the  quality  ordinarily  used  by  druggists. 
100  grammes  of  pyrogallic  acid. 

1  kilogramme  of  C.  P.  granulated  zinc. 

GLASSWARE. 

200  best  quality  test-tubes,  slightly  heavier  than  those 
used  for  chemical  work,  about  12  to  13  cm.  long  and  12 
to  14  mm.  inside  diameter. 

15  Petri  double  dishes  about  8  or  9  cm.  in  diameter 
and  from  1  to  1.5  cm.  deep. 

6  Florence  flasks,  Bohemian  glass,  1000  c.c.  capacity. 
6  Florence  flasks,  Bohemian  glass,  500  c.c.  capacity. 
12    Erlenmeyer    flasks,    Bohemian    glass,    100    c.c. 

capacity. 

1  graduated  measuring-cylinder,  1000  c.c.  capacity. 

1  graduated  measuring-cylinder,  100  c.c.  capacity. 

25  bottles,  125  c.c.  capacity,  narrow  necks  with 
ground-glass  stoppers. 

25  bottles,  125  c.c.  capacity,  wide  mouths,  with 
ground-glass  stoppers. 

1  anatomical  or  preserving  jar,  with  tightly  fitting 
cover,  of  about  4  litres  capacity,  for  collecting  blood- 
serum. 

2  battery  jars  of  about   2  litres  capacity,  provided 
with  loosely  fitting,  weighted,  wire-net  covers  for  mice. 


576  APPENDIX. 

10  feet  of  soft-glass  tubing,  2  or  3  mm.  inside  diam- 
eter. 

20  feet  of  soft-glass  tubing,  4  mm.  inside  diameter. 

6  glass  rods,  18  to  20  cm.  long  and  3  or  4  mm.  in 
diameter. 

6  pipettes  of  1  c.c.  each,  divided  into  tenths. 

2  pipettes  of  10  c.c.  each,  divided  into  cubic  centi- 
metres and  fractions. 

1  burette  of  50  c.c.  capacity,  divided  into  cubic  cen- 
timetres and  fractions. 

1  separating-funnel  of  750  c.c.  capacity  for  filling 
tubes. 

2  glass  funnels,  best  quality,  about  15  cm.  in  diam- 
eter. 

2  glass  funnels,  best  quality,  about  8  cm.  in  diameter. 

2  glass  funnels,  best  quality,  about  4  or  5  cm.  in 
diameter. 

2  porcelain  dishes,  200  c.c.  capacity. 

6  ordinary  water  tumblers  for  holding  test-tubes. 

1  ruled  plate  for  counting  colonies. 

1  gas-generator,  600  c.c.  capacity,  pattern  of  Kipp 
or  v.  Wartha. 


2  Bun  sen  burners,  single  flame. 
1  Rose-burner. 

1  Koch  safety-burner,  single  flame. 
6  feet  of  white-rubber  gas-tubing. 
12  feet  of  pure  red-rubber  tubing,  5  to  6  mm.  inside 
diameter. 

1  thermo-regulator,  pattern  of  L.  Meyer  or  Reichert. 

2  thermometers,  graduated  in  degrees  of  Centigrade, 
registering  from  0  to  100°  C.,  graduated  on  the  stem. 


APPENDIX.  577 

1  thermometer  graduated  in  tenths  and  registering 
from  0  to  50°  C. 

1  thermometer  registering  to  200°  C. 

INSTRUMENTS,    ETC. 

1  microtome,  pattern  of  Schanze,  with  knife. 

1  razor-strop. 

6  cheap-quality  scalpels,  assorted  sizes. 

2  pairs  heavy  dissecting- forceps. 

1  pair  medium-size  straight  scissors. 
1  pair  small-size  straight  scissors. 

1  hypodermic  syringe  that  will  stand  steam  steriliza- 
tion. 

2  teasing-needles. 

,  1  pair  long-handled  crucible-tongs  for  holding  mice. 

1  wire  mouse-holder. 

2  small  pine  boards  on  which  to  tack  animals  for 
autopsy. 

2  covered  stone  jars  for  disinfectants  and  for  receiv- 
ing infected  materials. 

INCUBATORS   AND   STERILIZERS. 

1  incubator,  simple  square  form,  either  entirely  of 
copper  or  of  galvanized  iron  with  copper  bottom. 

1  medium-size  hot-air  sterilizer  with  double  walls, 
asbestos  jacket,  and  movable  false  bottom  of  copper 
plates. 

1  medium-size  steam  sterilizer ;  either  the  pattern  of 
Koch  or  that  known  as  the  Arnold  steam  sterilizer, 
preferably  the  latter. 

MISCELLANEOUS. 

1  pair  of  balances,  capacity  1  kilogramme ;  accurate 
to  0.2  gramme. 

37 


578  APPENDIX. 

1  set  of  cork-borers. 
1  hand-lens. 

1  wooden  filter-stand. 

2  iron  stands  with  rings  and  clamps. 

3  round,  galvanized  iron-wire  baskets  to  fit  loosely 
into  steam  sterilizer. 

3  square,  galvanized  iron-wire  baskets  to  fit  loosely 
into  hot-air  sterilizer. 

1  sheet-iron  box  for  sterilizing  pipettes,  etc. 

1  covered  agate-ware  saucepan,  1200  c.c.  capacity. 

2  iron  tripods. 

1  yard  of  moderately  heavy  wire  gauze. 

2  test-tube  racks,  each  holding  24  tubes,  12  in  a  row. 

1  constant-level,  cast-iron  water-bath. 

2  potato-knives. 

2  test-tube  brushes  with  reed  or  wire  handles. 

Cotton-batting. 

Copper  wire,  wire  nippers. 

Round  and  triangular  files. 

Labels. 

Towels  and  sponges. 


INDEX. 


ABBE>  substage  condensing  sys- 
tem of,  30 
Abscess,  histological  study  of,  265- 

267 

production  of,  263,  264 
Aerobic  bacteria,  39 
Aerobioscope,  554 
Agar-agar,    preparation    of    (see 

Media). 

properties  of,  87,  88 
Agglutinin,  479 
Air,   bacteriological   analysis  of, 

551-556 

Petri's  method  for,  552 
Sedgwick-Tucker  meth- 
od, 553 
Alexins,  506 
Anaerobic  bacteria,  39 

methods  of  cultivating, 

206-212 
Buchner  s,  208 
Esmarch's,  211 
Frankel's,  208 
Hesse's,  206 
Kitasato  and  Weil's, 

211 

Koch's,  206 
Liborius's,  206 
Aniline  dyes   for  differentiating 

bacteria,  202 

Animals,   fluctuations   in   weight 
and  temperature  of,  233- 
238 
inoculation  of,  21 9 

apparatus  used  in,  221- 

223,  227,  230 
intralymphatic,  230 
intraocular,  233 
intraperitoneal  and  pleu- 
ral,  231 


Animals,  inoculation  of,  intravas- 

cular,  224 
subcutaneous,  219 
observations  of,  after  inocula- 
tion, 233 
post-mortem  examination  of, 

241-246 
cultures  from  tissues  at, 

243 

disinfection     of    imple- 
ments after,  245 
disposal  of  remains  from, 

245 

external  inspection,  241 
incision  through  skin, 

241 
Nuttall's  spear   for  use 

at,  244 

opening  the   body  cavi- 
ties, 242 

position  of  animal.  241 
precautions  during,  241, 

242 
preservation    of    tissues 

from,  244 
Anthrax,  448-462 

animals  susceptible  to,  445, 446 
bacillus  of,  448-456 

biology  of,  451-454 
discovery  of,  21;  448 
experiments  with,  458- 

462 

morphology  of,  448-451 
pathogenesis  of,  454-456 
protective  inoculation 

against,  456 
spore-formation,  449 
staining  of,  453 
symptomatic,  bacillus  of, 

482 

579 


580 


INDEX. 


Antiseptic,  definition  of,  82 
Antiseptics,  tests  of,  566 
Antitoxins,  496 

Apparatus  necessary  for  bacteri- 
ological work,  573 
preparation  of,  120 
Appendix,  list  of  apparatus,  573 


BACILLI,  45-48 
differentiation  from  spores,  48 
flagella  upon,  53 
involution-forms  of,  47 
life-cycle  of,  46 
mode  of  multiplication,  50- 

53 

motility  of,  53 
spore-formation  in,  47,  51 
Bacillus  anthracis,  448 
coli  communis,  393 
"comma,"  402 
diphtherise,  349 
Finkler-Prior,  429 
influenzas,  334 
leprse,  329 

mallei  (of  glanders),  341 
Neapolitans,  393 
nitrifying,  463 
oedematis  maligni,  476 
of  bubonic  plague,  292 
pyocyaneus,  287 
pseudo-diphtheria,  364 
smegma,  328 
subtilis,  257 

symptomatic  anthrax,  482 
syphilis,  328 
tetani,  469 
tuberculosis,  323 
typhosus,  369 
Bacteria,  aerobic,  39 
anaerobic,  39 

methods  of  cultivating, 

206-212 
special  method  of  Metch- 

nikoff,  Koux  and  Sal- 

embini,  248 
behavior  toward  staining-re- 

agents,  202 

capsule  surrounding,  163 
chromogenic,  34 
classification  of,  44 


Bacteria,  conditions  necessary  to 

growth  of,  31-43 
constancy  in  morphology  of, 

47 

definition  of,  31 
denitrifying,  35 
discovery  of,  17-19 
facultative,  40 
fermentation  by,  203 

apparatus  for  testing,  204 
gases  resulting  from ,  205 
flagellated  forms  of,  53 
identification  of,  189 
involution-forms  of,  47 
isolation  of,  in  pure  culture, 

83-89 
on  slanted   media,   134, 

135 

principles  of,  83-87 
microscopic  examination  of, 

190-200 
modes   of  multiplication  of, 

49-52 

morphology  of,  44-54 
motility  of,  53 
nitrifying,  35,  463 
nutrition  of,  36-40 
pathogenic,  33,  35 
photogenic,  34 

points  to  be  observed  in  de- 
scribing, 216 

reaction  produced  by,  201 
relation  to  man,  32,  33 
to  temperature,  41 
results  of  growth,  33-36 
role  in  nature,  32 
saprogenic,  35 
spore-formal  ion  of,  47,  51 

study  of,  197 
staining-reactions  of,  202 
systematic  study  of,  189 
thermophilic,  41 
thiogenic,  35 

very  minute,  method  of  ex- 
amination of,  248,  249 
zymogenic,  34 

Bacteriology,  application  of  meth- 
ods of,  251 

Bacterium  coli  commune,  393-400 
characteristics  of,  cul- 
tural, 395-398 


INDEX. 


581 


Bacterium   coli   commune,  char- 
acteristics of, 
m  o  rphologi- 
cal,  394 
pathogenic,  398 
differentiation      of, 
from   bacillus  ty- 
phosus,  397 
where  found,  393 
Behring  and  Kitasato,  509 
Billroth,  27 

and  Tiegel,  28 
Birch-Hirechfeld,  26 
Black  leg  (see  Symptomatic  An- 
thrax). 
Blood,  relations  to  bacteria  and 

to  toxins,  502,  509 
Blood-serum   as  culture-medium 

(see  Media). 

germicidal  element  of,  506 
Bol ton's  potato  method,  104 
Bonnet,  '24 
Booker's    modification     of     Es- 

marcli's  method,  132 
Bouillon  (see  Media). 
Brieger  and  Cohn,  476 
Brooding-oven,  136-138 
Brcwnian  motion,  196 
Buchner,  506,  510 
Bulb  for  water  samples,  540 
Burdon-Sanderson,  29 
Burner,    Koch's    safety,   for    use 
with  incubator,  138 


/CARBOLIC  acid  as  disinfectant, 

\J     80 

Chauveau,  498 

Cheraotaxis,  42 

(  hevreul  and  Pasteur,  23 

Chlorophyll,  31,  32 

Cholera    Asiatica,    diagnosis    of, 

423-428 
method  of  Schotte- 

lius,  410 
spirillum  of,  402 

behavior  of,  in  but- 
ter, 421 
in  milk,  420 
in  soil,  419 
in  water,  417 


Cholera    Asiatica,    spirillum   of, 

characteristics 

of,      cultural, 

404 

morphological, 

effects  of  dry  ing,  422 
existence  outside  the 

body,  417 

experiments     upon 
animals  with,  412 
general     considera- 
tions upon,  416 
isolation  of,  423 
location  in  the  body, 

416 
persistency     of,     in 

dead  body,  422 
poisons  produced  by, 

411 
relation     to    gases, 

409,  422 
to  other  bacte- 
ria, 410,  421 
to  putrefaction, 

420,  421 
to  sunlight,  419 
specific  reaction  of 
immuned  animals 
to,  415,  416 
toxin  of,  411 
Chromogenic  bacteria,  34 
Classen,  27 
Cohn,  25 
Colon  bacillus  (see  Bacterium  Coli 

Commune). 
Colonies,  counting  of,  547 

study  of,  145 
Comma     bacillus     (see     Cholera 

Asiatica). 
Cornet,  321 

Corrosive  sublimate  as  disinfect- 
ant, 75,  76 

Cooling-stage,  127,  131 
Cover-slips,  cleaning  of,  151 
impression,  155 
microscopic  examination  of, 

193 

preparation  of,  151 
steps  in  making,  152 
Cultures,  gelatin,  199 


582 


INDEX. 


Cultures,  hanging-drop,  195 

potato,  200 

pure,  146 

reactions  of,  201 

stab-  and  smear-,  146 
Cygnseus,  375 


r\ECOLOKIZING       solutions, 
I)     173 

Decomposition,  31,  463 
Defensive  proteids,  507 
Deneke's  cheese  spirillum  (see 

Spirillum,  tyrogenum). 
Denitrifying  bacteria,  35 
Diphtheria,  bacillus  of,  349-368 
cultural  peculiarities  of, 

355-358 

experiments  upon,  340 
location  in  tissues,  358- 

361 

method  of  obtaining,  349 
modification     in     viru- 
lence, 362-365 
morphology  of,  350-354 
pathogenesis  of,  358-362 
poison  produced  by,  362 
principles   of   immuniz- 
ing against,  509 
pseudo-diphtheria  bacil- 
lus, 364 

histological  changes  accom- 
panying, 360-362 
Diplococci,  46 

Disinfectants  and  antiseptics,  ex- 
periments with,  567 
general  considerations,  73-82 
methods  of  testing,  558 

precautions    to    be    ob- 
served, 561 
use  of  animals  as  test-objects 

for,  565 

use  of,  in  the  laboratory,  80 
Disinfection,    general    considera- 
tions, 73-80 
influence  of  temperature  on, 

76 

inorganic  salts  in,  74-79 
in  the  laboratory,  80 
investigations  of  Kronig  and 
Paul  on,  77 


Disinfection,  modus  operandi.  75- 

79 
reliable  agents  for  purposes 

of,  80,  81 
selection  of  agents  to  be  used 

in,  74 
Dunham's  solution,  115 


EBEKTH,  27 
Ehrlich,  27 

"side-chain"  theory  of 

immunity  of,  518 
Eisner's  medium,  384 
Emmerich  and  Fowitzky,  478 

and  Mattei,  472 
Enzymes,  34 
Erysipelas,  272 
Escherich,  393 
Esmarch  tubes,  131 

Booker's  method  of  roll- 
ing, 132 

made  of  agar-agar,  133 
Examinations,        bacteriological, 

during  life,  246 

Exposure      and     contact-experi- 
ments, 253 


TUCULT  ATI  VE  bacteria,  31, 40 
Jj      Fehleisen,  27 
Fermentation,  34,  203 

gases  resulting  from,  205 
particular  forms  of,  34-36 
-tube,  204 

method  of  using,  204,  205 
Filter,  method  of  folding,  96,  97 
Finkler-Prior  bacillus,  429 
Flagella,  53 

methods  of  staining,  167-171 
Bunge's,  169 
Loffler's,  167 
van  Ermengem's,  170 
Flagellated  organisms,  53 
Fluids,    examination    of,  during 

life,  246 

Fowl  tuberculosis,  332 
Frankland,  G.  and  P.  F.,  465 
Funnel   for   filling   aerobioscope, 

510 
for  filling  test-tubes,  122 


INDEX. 


583 


Funnel  for  filtering  cultures,  517 
hot-water,  98 


p  AS-PRESSURE  regulator,  142 
VJ     Gelatin,  cultures  in,  199 

their         characteristics, 

199,  200 

preparation  of  (see  Media), 
properties,  87,  88 
Geppert,  76 
Glanders,  339-348 
bacillus  of,  341 

cultivation  of,  343-347 
inoculation  with,  344 
morphology  of,  341 
staining  of^  in  tissues,  345 
diagnosis  of,  by  use  of  mal- 

lein,  348 

by  Strauss's  method,  347 
manifestations  of,  339,  340 
histology  of,  340,  341 
susceptibility  of  animals  to, 

344 

synonyms,  339 
Gonococcus,  277-287 

appearance  in  pus,  277 
cultivation  of,  279-284 
Bumm's  method,  279 
Wassermann's     method, 

282 

Wertheim's  method,  279 
Wright's  method,  280 
distinguishing  features  of,  286 
morphology  of,  278 
organisms  that  simulate,  285 
pathogenesis  of,  284-286 
vitality  of,  284 
Gonorrhoea,  pus  of,  277 
Green  pus  bacillus  (see  Bacillus 

pyocyaneus). 
Guarniari's  agar-gelatin,  118 


HALSTED,  231-263 
Hanging-drop,  195 
Hankin,  506,  507 

and  Martin,  506 
Henle,  22 

Hiss's  medium,  387 
Hoffmann,  23 


Hot-water  funnel,  98 
Hydrogen,  test  for  purity  of,  210 
Hypodermic  syringes  and  needles, 
227,  230 


IMBEDDING    of   tissues,   176, 
1     177 

Immunity,  497 
acquired,  498 

blood  in,  502 

conclusions  concerning,  522 
earlier  studies  on  blood  rela- 
tive to,  503-505 
Ehrlich's  theory  of,  518 
"exhaustion"  hypothesis,  500 
experiments   of   the    Klem- 

perers  on,  514 
hypothesis  of  Buchner,  517 
evidence  in  favor  of, 

512-514 
natural,  497 
nature  of  protective  bodies, 

494,  495,  506,  507,  520 
observations  of  Behring  and 

Kitasato,  509 

"retention"  hypothesis,  499 
theory  of  Metchnikoff,  501 
Incubator,  136 

burner  for  heating,  138 
Indol,  method  of  detecting,  213 
production    of,    by   bacteria, 

213 
Infection,  489-493 

chemical  nature  of,  493 
conclusions  concerning,  522 
modus  operandi,  495 
poisons  present  in,  494 
study  of  types  of,  489-491 
Influenza,  baci'llus  of,  334-338 
cultivation  of,  335-337 
dissemination  of,  337 
isolation  of,  from  tissues, 

336,  337 

morphology  of,  335 
occurrence  of,  in  tissues. 

337 

staining  of,  335 
susceptibility  of  animals 

to,  338 
vitality  of,  337 


584 


INDEX. 


Inoculation  of  animals,  219-233 
apparatus  used  in,  221, 

223,  227,  230 
intraocular,  233 
intraperitoneal  and  pleu- 

ral,  231 

intralymphatic,  230 
intravascular,  224 
subcutaneous,  223 
Involution-forms  of  bacteria,  47 
Isolation  of  colonies  on   slanted 
media  in  tubes,  134 


J 


ORDAN  and  Richards,  465 


KLEBS,  27,  351 
Klemperer,  F.  and  G.,  work 

on  pneumonia,  475 
Koch,  fundamental  researches  of, 

29 

postulates  of,  323 
safety  burner  of,  138 
Kronig  and  Paul,  77 


ACTOSE-LITMUS    agar-agar 

or  gelatin  (see  Media). 
Leeuwenhoek,  17-19 
Lens      for      counting      colonies, 

548 
Lepra  bacillus,  329 

staining-peculiarities  of, 

330 

Letzerich,  27 
Levelling-tripod,  127 
Lime,  chloride  of,  81 

milk  of,  80 
Litmus  milk,  114 
Loffler's  alkaline  methylene-blue, 

158 

blood-serum  mixture,  118 
method  for  the  isolation  of 
the  bacillus  of  diphtheria, 
349 

stain  for  flagella,  54,  167 
Loffler  and  Schiitz,  discovery  of 

the  bacillus  of  glanders,  341 
Lukomsky,  27  • 


MALIGNANT  oedema,  bacillus 
Ifl  of,  476-481 

cultural     peculiari- 
ties of,  478,  479 
morphology  of,  478 
pathogenesis  of,  479 
susceptibility  of  ani- 
mals to,  480 
Mallein,  348 
Meat-extracts  in  culture-media,  95 

-infusion,  119 
Media,  culture,  90 

agar-agar,  100 

clarification  of,  101 
filtration  of,  101 
glycerin,  102 
neutralization       of, 

90-95,  103 
solution  of,  101,  102 
blood-serum,  106 

Councilman  -  Mai- 
lory  method,  110 
mixture  of  Loffler, 

118 
Nuttall's      method, 

111 
original  method  of 

Koch,  106 
preservation  of,  1 1 0- 

113 
by   chloroform, 

113 

sterilization  and  so- 
lidification        of, 
108-110 
bouillon,  90 

•  neutralization       of, 

90-95 
Eisner's     potato-gelatin, 

384 
gelatin,  95 

clarification  of,  98 
filtration  of,  96-98 
solution  of,  96 
sterilization  of,  99 
Guarniari's       agar-agar 

gelatin,  118 
Hiss's  medium,  387 
lactose-litmus    agar-agar 

or  gelatin,  117 
litmus  milk,  114 


INDEX. 


585 


Media,  culture,  meat-infusion,  119 
milk,  114 

-agar-agar,  114 
peptone   solution,    Dun- 
ham's, 115 
Proskauer  and 
Capaldi's,  390 
rosolic-acid-peptone    so- 
lution, 116 
potatoes,  103 

Bol  ton's       method, 

104 
Esmarch's    method, 

105 

mashed,  105 
original  method,  103 
Stoddard's  medium,  386 
Metchnikoff,  463 

Roux  and  Salembini,  method 

of,  248 

Milk  (see  Media). 
Micrococci,  44,  49 

mode  of  multiplication  of,  49 
Micrococcus  lanceolatus,  303-308   i 
irregularities"  in    devel-  : 

opment  of,  307 
morphological   peculiar- 
ities of,  300,  304,  305    \ 
results     of     inoculation 

with,  307 
staining  of,  307 
susceptibility  of  animals 

to,  308 
variations    in    virulence 

of,  307 

where  found,  305 
tetragenus,  302,  309-311 

cultural  peculiarities  of, 

309-311 

morphology  of,  309 
susceptibility  of  animals 

to,  311 

where  found,  309 
Microscope,  parts  of,  190 
Microtome,  175 


NAGELI,  37 
Nassiloft;  27 
Needham,  22 
Nitrification,  463 


Nitrifying  bacteria,  428-433 
Nitrites,  test  for,  215 
Nitro-monas     of     Winogradsky, 
465-468 

cultural  peculiarities  of, 
466-408 

morphology  of,  466 
Nocard  and  Roux,  247 
Normal  solution,  208 
Nuttall,  243,  295,  296,  502 


OERTEL,  27,  361 
Oil   immersion   system,  use 

of,  192,  194 
Ozanam,  21 


DARASITE,  31,  32 
I       Parietti's  solution,  537 
Pasteur,  21,  23,  29,  39,  500 
Peptone,  test  of  purity   of,   115, 

116 

with  rosolic  acid,  116 
Peritonitis,  production  of,  263 
Petri's  dishes,  130 
Pfeiffer,  415,  416,  513,  521 
Phagocytosis,  501 
Photogenic  bacteria,  34 
Plague,  bubonic,  bacillus  of,  292- 

299 

cultivation  of,  294,  295 
mode  of  infection  with, 

296-299 

morphology  of,  294 
occurrence    in     tissues, 

294-296 

pathogenesis  of,  295,  296 
vitality  of,  295 
Plates,    apparatus    employed    in 

making,  124-129 
Esmarch's  modification,  131 
Booker's  modification  of, 

132 

Koch's    fundamental    obser- 
vations, 83-87 
materials    used    in    making, 

124 

Petri's  modification,  130 
principles    involved    in   the 
method,  83-87 


586 


INDEX. 


Plates,  technique  of  making,  124- 

129 

Platinum  needles  and  loops,  125 
Plenciz,  20 

Pleuro-pneumonia  of  cattle,  247 
Post-mortem  examination  of  ani- 
mals, 241-246 
cultures  from  tissues  at, 

243 

disinfection     of     imple- 
ments after,  245 
disposal  of  remains  from, 

245 
external    inspection    at, 

241 
incision      through     the 

skin  at,  241 
Nuttall's    spear  for  use 

at,  243 

opening  of  the  body-cav- 
ities, 242 

position  of  animal  dur- 
ing, 241 
precautions  during,  241, 

242 

preparation     of     cover- 
slips  at,  244 
preservation     of    mate- 
rials after,  244 
Postulates  of  Koch,  323 
Potato,  characteristics  of  cultures 

on,  200 
preparation    of,    for    culture 

purposes  (see  Media). 
Proskauer  and  Capaldi,  390 
Prudden,  317,  467 
Pseudo-diphtheria  bacillus,  364 

-tuberculosis,  334 
Ptomains,  36 
Pure  culture,  146 
Pus,  microscopic  appearance   of, 

260,  268,  277 
Putrefaction,  31 
Pyaemia,  production  of,  264 
Pyocyaneus,  bacillus,  287-291 

chameleon     phenomena 

of,  289 
pathogenic  properties  of, 

290 

protective  properties  of, 
291 


QUARTER  evil  or  quarter  ill 
(see  Symptomatic  Anthrax). 


DECKLINGHAUSEN,  26,  27 
J\     Regulator,  gas-pressure,  142 

therrno-,  139 
Rindfleisch,  26 
Rosolic-acid-peptone  solution  (see 

Media). 
Roux  and  Yersin,  303 


01 APROGENIC  bacteria,  35 
O     Saprophyte,  31 

role  of,  in  nature,  32 
Sarcinee,  46 

mode  of  multiplication.  50 
Schottelius's  method  of  examin- 
ing cholera  evacuations,  410 
Schroder  and  Dusch,  23 
Schulze,  23 
Schwaun,  23 
Section-cutting,  175 
Septicaemia,  302 

from  micrococcus  tetragenus, 

308 

from  sputum,  303 
"Side-chain"  theory  (Kettenthe- 

orie)  of  Ehrlich^  518 
Skin-disinfection,  experiments  in, 

570 

Smear-cultures,  146 
Srnegrna   bacillus,   staining-pecu- 

liarities  of,  328,  329 
Soil,  bacteriological   analysis   of, 

556 

nitrifying  bacteria  in,  463 
organisms  present  in,  468 
phenomena  in  operation  in, 

463 

Spallanzani,  22,  23 
Spirilla,  46,  49 
Spirillum  of  Asiatic  cholera  (see 

Cholera). 

of  Deneke,  434-437 
biology  of,  434-437 
morphology  of,  434 
pathogenesis  of,  437 
of  Finkler-Prior  (see  Vibrio 
Proteus). 


INDEX. 


587 


Spirillum    of    Metchnikoff    (see 

Vibrio  Metchnikovi). 
of  Miller,  437-441 

biology  of,  438-440 

morphology  of,  438 

pathogenesis  of,  441 

tyrogenum  (see  Spirillum  of 

Deneke). 
undula,  53 
Spores,  formation  of,  46,  51 

method  of  studying,  46, 

48,51,52,  197 
mode    of   development,   46- 

52 

recognition  of,  46-48 
staining  of,  164-167 
Sputum,  inoculations  with,  302 
microscopic  examination  of, 

300,  301 
pathogenic  properties  of,  302, 

303 

septicaemias,  303-308 
tuberculosis,  303-312 
tuberculous,  300 
Stab-cultures,  146 
Staining,  methods  and  solutions 

used  in,  150-188 
acetic  acid,  163 
Bowhill's,  171 
Bunge's,  169 
Gabbett's,  162 
general  remarks  on,  172 
Gram's,  162 
Gray's,  185 
Koch-Ehrlich's,  158 
Kuehne's,  183 
Loffler's  blue,  158 

flagella,  167 
Moeller's,  165 

ordinary  solutions  used,  156 
bottles  for  hold- 
ing, 157 

van  Ermengem's,  170 
Weigert's  183 
Ziehl-Neelsen,  159, 185 
Staphylococcus    pyogenes    albus. 

267 
aureus,  260-267 

cultural     peculiari- 
ties of,  261,  262 
pathogenesis  of,  263 


Staphylococcus  pyogenes  aureus, 
where  to  be  expected, 
262 

citreus,  267 
epidermidis  albus,  268 
Sterilization,  chemical,  74 
by  heat,  57 

principles  involved,  59- 

64 
by  hot  air,  58 

apparatus  used,  72 
by  steam,  58-71 

apparatus  used,  67-71 
under  pressure,  65-69 
direct,  65 

experiments  upon,  255 
intermittent,  60-65 

at  low  temperature,  64 
principles  involved,  60-65 
use  of  the  term,  55-57 
Sternberg,  305 
Stoddard's  medium,  386 
Strauss's  method  for  diagnosis  of 

glanders,  347 
Streptococci,  46 

mode  of  multiplication,  50 
Streptococcus  pyogenes,  268-274 
biology  of,  270-272 
effects     of     inoculation 

with,  273 

morphology  of,  268,  269 
where  to    be    expected, 

268,  275 

Subtilis  bacillus,  257 
Suppuration,  260 

bacteria    common     to,    267, 

276 

general  remarks  upon,  276 
less  common  causes  of,  267, 

276 
microscopic    appearance    of 

pus,  260 
Symbiosis,  42 
Symptomatic  anthrax,  bacillus  of, 

482-488 

biology  of,  483-486 
differentiation  from   ba- 
cillus   of     malignant 
o?dema,  487 
morphology  of,  482 
pathogenesis  of,  486 


588 


INDEX. 


Symptomatic  anthrax, susceptibil- 
ity of  animals  to,  487 

Syphilis  bacillus,  staining  of,  328- 
330 


T1ECHNiQUE,  novel,  248 
1     Test-tubes,  cleaner  for,  120 
cleaning  of,  120 
filling  with  media,  121 
apparatus  for,  122 
plugging  with  cotton,  121 
position  after  filling,  123 
sterilization  of,  123 
Tetanus,  bacillus  of,  469-476 
biology  of,  471 
effects  on  animals,  474 
method  of  obtaining,  469 
morphology  of,  471 
poison  produced  by,  475, 

476 

toxin,  potency  of,  476 
specific  affinity  for  nerve- 
cells,  519 
Tetrads,  46 

Thermophilic  bacteria,  41,  63 
Therrno-regulator,  139 
Thermostat  (see  Incubator). 
Thiogenic  bacteria,  35 
Tissues,  cultures  from,  at  autop- 
sies, 243 
examination  during  life, 

246 

Nuttall's  spear  for  mak- 
ing, 243 

cutting  sections  of,  175 
hardening  of,  174 
imbedding  of,  176 
in  celloidin,  176 
in  paraffin,  177 
preservation  of,  174 
staining  bacteria  in,  174-188 
special  methods,  181-188 
dahlia,  182 
dry,  185 
Ehrlich's,  185 
Gram's  181 
Gray's,  185 
Kuehne's  183 
Weigert's  183 
^  Ziehl-Neelsen's,  185 


Tissues,  staining  bacteria  in,  steps 

in  the  process,  180 
Toxaemia,  492 
Toxalbumins,  36 
Toxins,  36,  494 
Traube  and  Gscheidlen,  503 
Treviranus,  23 

Tripod  for  levelling  plates,  127 
Tube,  Esmarch,  131 
Tuberculin,  331 
Tuberculosis,  312-323 
aviau,  332 

cavity-formation  in,  316 
conditions    simulating,  332- 

334 

diffuse  caseation  of,  315 
encapsulation   of    tubercular 

foci,  317 

giant  cells  in,  315 
location  of  bacilli  in,  321 
manifestations  in  experimen- 
tal, 313 
miliary     tubercle,    structure 

of,  314 

modes  of  infection,  319 
primary  infection,  318 
pseudo,  334 
sputum  in,  300 

inoculation    of    animals 

Avith,  302 
microscopic    appearance 

of,  300,  301 
staining  of,  160 
susceptibility  of  animals  to, 

332 

Tuberculosis,  bacillus  of,  323-334 
appearance   of  cultures, 

325 
cultivation  from  tissues, 

324 
methods  of  staining,  160, 

185-188 

dry  method,  185 
Gabbett's,  162 
Gray's,  185 
Koch    -    Ehrlich's, 

158,  185 

Nuttall's     modifica- 
tion, 161 

Ziehl     -    Neelsen's, 

159,  185 


INDEX. 


589 


Tuberculosis,  bacillus  of,  micro- 
scopic appearance  of, 
326 

organisms  that  simulate 
it,  328 

differential   diagno- 
sis of,  328-330 
staining    of,  in    tissues, 

185-188 
staining-peculiarities  of, 

327 

toxin  of,  494 
Tyndall,  24 
Typhoid  fever,  bacillus  of,  369- 

392 
constant    properties 

of,  379 
cultivation  of,  370- 

374 

difficulty  in  identi- 
fying," 378 

differentiation  from 

bacillus    coli 

communis, 

397 

Hiss's  method, 

387 

Parietti's  meth- 
od, 537 

Proskauer  and 

Capaldi's 

method,  390 

Stoddard's 

method,  386 

Eisner  medium  for 

isolating,  384 
experiments     with, 

392 
inoculations      with, 

374 

location   of,   in   tis- 
sues, 373 

morphology  of,  369 
reaction  of,  with  ty- 
phoid serum,  379 
source   from   which 

to  obtain,  391 
water  as  a  carrier  of, 

526 

Widal's        reaction 
with,  380 


TTAUGHAN,  507 
Y      Vibrio    Metchnikovi,    441- 

446 

characteristics  of,  cultu- 
ral, 441-444 
morphological,  441 
pathogenesis  of,  444-446 
Vibrio  proteus  of    Finkler   and 

Prior,  429-434 
cultivation  of,  430 
morphology  of,  429 
pathogenesis  of,  433 
relation  to  cholera  nos- 

tras,  429,  434 
Schuylkilliensis,  446 
Vibrion  septique,  476 


WALDEYER,  26 
Wassermann,    studies    on 

tetanus  toxin,  519 
Water,  general  observations  upon 
bacteriological    study    of, 
526-532 
qualitative       bacteriological 

analysis  of,  532 
precautions    in    ob- 
taining    sample, 
533 
preliminary      steps 

in,  533 
quantitative     bacteriological 

analysis  of,  538 
collection  of  sample, 

539-542 

counting  colonies  in, 
546 


apparatus 
547-550 


for, 


selection   of  proper 
medium  for,  544- 
546 
relation     of,    to    epidemics, 

526 
typhoid  bacilli  in,  529,  530, 

537 

value  of  bacteriological  ex- 
amination of,  530 
value  of  chemical  examina- 
tion of,  528 
Weigert,  30,  518 


590 


INDEX. 


Weigert,  doctrine  of  cell-equilib- 
rium of,  518 

Welch,  276,  303,  306 

WidaPs  reaction,  380 

Wilde,  27 

Wlnogradsky,  nitro-monas  of, 
465 


Wound-infection,  26-29 


Wurtz's 
117 


agar-agar    and    gelatin, 


ZOOGLGEA  of  bacteria,  48 
Zymogenic  bacteria,  34 


- 


WAY  10 


LIBRARY 
6    . 


UNIVERSITY  OF  CALIFORNIA  LIBRARY 


